Compounds and methods for regulating  integrins

ABSTRACT

A method of treating inflammation, by administering an effective amount of a β2 integrin agonist to a patient, and reducing inflammation. A method of treating cancer, by administering an effective amount of a β2 integrin agonist to a patient, and reducing tumor growth. A method of treating a patient exposed to radiation, by administering an effective amount of a β2 integrin agonist to the patient after radiation exposure, and mitigating the effects of radiation exposure in the patient. A method of preventing effects of radiation, by administering an effective amount of a β2 integrin agonist to the patient prior to radiation exposure, and preventing the effects of radiation exposure on the patient. A method of treating acquired bone marrow failure (BMF), by administering an effective amount of a β2 integrin agonist to a patient. Methods of improving the health of damaged vasculature in a patient and activating β2 integrins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/517,667, filed Oct. 17, 2014, which is a continuation ofPCT/US2013/037548, filed Apr. 22, 2013, which application claims thebenefit of priority to U.S. Provisional Application Ser. No. 61/635,968filed Apr. 20, 2012, and to U.S. Provisional Application Ser. No.61/791,523, filed Mar. 15, 2013, which are each incorporated herein byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to priming or activation of β2 (beta2)family of integrins with various agents. The present invention furtherrelates to treating various diseases and conditions that involve beta2family of integrins.

2. Background Art

Integrins are non-covalently linked α/β heterodimeric receptors thatmediate cell adhesion, migration and signaling. Together with theirligands, integrins play central roles in many processes includingdevelopment, hemostasis, inflammation and immunity, and in pathologicconditions such as cancer invasion and cardiovascular disease. Keyleukocyte functions, such as activation, migration, tissue recruitmentand phagocytosis, are essential for their normal immune response toinjury and infection and in various conditions, including inflammatoryand autoimmune disorders [1, 2]. The β2 (b2) integrins, a sub-family ofα/β heterodimeric integrin receptors have a common β-subunit (β2, CD18)but distinct α-subunits (CD11a, CD11b, CD11c and CD11d [3]) [4]. Theyregulate leukocyte functions, including via highly expressed integrinsCD11a/CD18 (also known as LFA-1) and CD11b/CD18 (also known as Mac-1,CR3 and αMβ2) [2] that recognize a variety of ligands. For example,CD11b/CD18 recognizes >30 ligands, including the complement fragmentiC3b, Fibrinogen, CD40L and ICAM-1 as ligands, among various others.CD11b/CD18 has been implicated in many inflammatory and autoimmunediseases. These include ischemia-reperfusion injury (including acuterenal failure and atherosclerosis), Multiple Sclerosis (MS), tissuedamage, transplantation, lupus, lupus nephritis, macular degeneration,glaucoma, stroke, neointimal thickening in response to vascular injuryand the resolution of inflammatory processes [5-9]. For example,leukocyte infiltration and plaques of demyelination in the brain andspinal cord of patients are a hallmark of MS and CD11b/CD18 has beenshown to play a key role in mediating leukocyte adhesion, migration andtrafficking in MS and is a validated target for MS. Similarly, influx ofinflammatory leukocytes potentiates anti-GBM nephritis, which is a modelof rapidly progressive glomerulonephritis and lupus nephritis, and ischaracterized by proteinuria, leukocyte infiltration and glomerularcrescent formation [10, 11]. Leukocytes play a critical role in thepathogenesis of anti-GBM nephritis, and their number correlates with thepercentage of crescentic glomeruli. CD11b^(−/−) animals show noproteinuria and strong protection of renal function [12], suggestingthat agents targeting this integrin have a potential to treat thisdisease.

According to the American Cancer Society, worldwide, nearly 8 M peopledie from cancer every year. This number is expected to rise to 13.1 Mdeaths per year by the year 2030. There were 13.2 M new cases of cancerin the world in 2008, with an associated cost burden of $290 B, andthese cases are expected to rise to 22.2 M by 2030, with a cost burdenof $458 B. The developing world sees twice as many new cases of canceras the developed world. Cancer is the second most common cause of deathin the US; nearly 600,000 Americans are expected to die of cancer in2013, almost 1,600 people per day, accounting for nearly 1 of every 4deaths. About 1.6 M new cancer cases are expected to be diagnosed in2013.

Breast cancer (BC) is the second most common cancer among women in theUS; 1 in 8 women will have BC in their lifetime; BC is also a leadingcause of cancer death among women of all races; ˜226,000 new cases ofinvasive BC in 2012; almost 40,000 women die from BC every year. Besidesbeing female, age is the most important risk factor for BC. BC producesno symptoms when the tumor size is small and large tumors may becomeevident as a breast mass, but are also often painless. Breast pain ismore likely to be caused by benign conditions and is not a common earlysymptom of BC.

Currently, surgical removal of part or whole breast is the mosteffective treatment for early-stage BC, in combination with radio- andchemo-therapy. Postmenopausal women with early stage breast cancer thattests positive for hormone receptors benefit from treatment with anaromatase inhibitor (e.g., letrozole, anastrozole, or exemestane) inaddition to, or instead of, tamoxifen. For women whose cancer testspositive for HER2/neu, approved targeted therapies include trastuzumab(Herceptin) and, for advanced disease, lapatinib (Tykerb) and pertuzumab(Perjetal). The US Food and Drug Administration (FDA) revoked approvalof bevacizumab (Avastin) for the treatment of metastatic breast cancerin 2011 because of evidence showing minimal benefit and some potentiallydangerous side effects. Thus, additional therapeutics that are moreeffective and have fewer side effects are greatly needed. Furthermore,adjuvant therapeutics that can significantly reduce the dose of toxicchemo- and radio-therapeutic regimens in patients with BC are greatlyneeded.

Also, a majority of currently used anticancer therapies have significantcardiovascular safety concerns. Dose-dependent and progressive leftventricular (LV) dysfunction manifesting as symptomatic heart failure iswell documented in patients receiving anthracyclines. In women withearly breast cancer, particularly those >65 years of age, cardiovasculardisease (CVD) is now the most common cause of death as indicated bySurveillance, Epidemiology, and End Results (SEER)-Medicare linked data.Additionally, these women are also at increased risk of CVD comparedwith age-matched women without a history of breast cancer. Paclitaxel isarrythmogenic cytotoxic drug and leads to bradycardia, with incidencerate with paclitaxel ranging from 0.5% to 5% (and 1.7% with docetaxel).While the main cardiotoxicity of taxanes is bradycardia, ischaemia hasalso been described. Importantly, clinical trials with the newertherapeutics, such as human epidermal growth factor receptor 2(HER2)-directed monoclonal antibodies (i.e. trastuzumab) and other newermulti-targeted small-molecule inhibitors show that interfere withmolecular pathways crucial to normal cardiac homeostasis, resulting inrelatively high incidences of subclinical and overt cardiac toxicity.Even more significantly, while the cardiac toxicity with newer therapiesmay be reversible, the recovery. of LV function after treatmentcessation is uncertain at this time. Trastuzumab (Herceptin, a humanizedmonoclonal antibody against the HER2 tyrosine kinase receptor) shows theincidence of LVEF decrease or asymptomatic heart failure (HF) by ˜7%,but it can rise to 13% when trastuzumab is administered with concurrentpaclitaxel and to 27% with concurrent anthracyclines. Thus, there is agreat need for newer therapeutics for BC, which, in addition to beingmore efficacious, also lower the cardiovascular risk.

Inflammatory Leukocytes Recruited to Tumor Microenvironment are Targetsfor Cancer Therapy. Inflammation is directly linked to rumor growth andre-growth post treatment with surgery, anti-cancer agents and radiation.CD45+ leukocytes are significantly upregulated in naive human breasttumors and after chemo-therapy. Myeloid cells (e.g.; neutrophils andmacrophages) are among the cell types that are highly upregulated in thetumors, especially post treatment. In multiple animal models, reducinginfiltration of myeloid cells leads to significant reduction in tumorburden, improves efficacy of cancer therapies and reduces BC metastasis.For example, it was recently shown that anti-CD11b antibodies enhancetumor response to radiation in models of squamous cell carcinoma.

Leukocytic β2 integrins also modulate tumor infiltration. For example,tumors also secrete inflammatory cytokines to recruit CD11b-expressingmyeloid cells to facilitate neovascularization [13]. During cancertreatments, irradiated tumors recruit large numbers of specificleukocytes, such as bone marrow-derived CD11b-expressing myeloid cellsexpressing matrix metalloproteinase-9 (MMP-9), that restore tumorvasculature and allow tumor re-growth and recurrence [14]. Recentstudies have shown that treatment with CD11b antagonists (anti-CD11bantibody) reduces CD11b-expressing myeloid cell infiltration and anenhancement of tumor response to radiation in mice [14], suggesting thatagents targeting this integrin have a potential to be used astherapeutics in oncology.

Additionally, exposure to ionizing radiation (IR) causes injury inanimals, eliciting an influx of inflammatory leukocytes that is partlyresponsible for early (acute) and late (chronic) injury and progressivefunctional impairment of multiple critical organs in mammals [15-20].These include the hematopoietic system. The consequences of exposure toionizing radiation (IR) are of major concern for patients that have, forexample, undergone radiation therapy and individuals that are exposed toIR due to nuclear accident or attack. Moreover, exposure to sublethal IRalso causes dose-dependent injury, including the hematological toxicityand also affects both the hematopoietic stem cell (HSC) numbers andtheir function (functional damage), including their capacity forlong-term repopulation [21-26]. Therefore, blockage or reduction ofinflammatory responses after radiation exposure could help mitigateearly (acute) and late (chronic) effects of radiation in exposedpatients.

Furthermore, acquired bone marrow failure (BMF) develops after an injuryto the bone marrow (BM) by ionizing radiation (IR), chemotherapy drugsand antibiotics (e.g. busulfan and chloramphenicol), toxic chemicals(benzene, carbon tetrachloride), or viral infection (hepatitis, HIV,CMV, parvovirus). Another form of acquired BMF called aplastic anemia isan immune-mediated BMF that develops after lymphocyte infusion, and ischaracterized by an immune-mediated functional impairment ofhematopoietic stem cells (HSCs). Functional damage in HSCs can over timelead to development of acquired BMF.

CD11b/CD18 is also expressed on short-term repopulating hematopoieticstem cells (HSCs) and hematopoietic progenitors (HPCs), and has beenshown to participate in the retention and anchoring of HPCs in the bonemarrow during enforced mobilization, suggesting that agents targetingCD11b/CD18 can have a protective effect on the number and function ofHSCs and HPCs, in vitro, ex vivo, and in vivo.

Studies over the last several years have shown that blocking CD11b/CD18and its ligands with antibodies and ligand mimics (anti-adhesiontherapy) [24-26] and genetic ablation of CD11b or CD18 decreases theseverity of inflammatory response in vivo in many experimental models[27, 28]. However, such blocking agents have had little success intreating inflammatory/autoimmune diseases in humans [28, 29], perhapsbecause complete blockage of CD11b/CD18 with antibodies is difficult dueto availability of a large mobilizable intracellular pool of CD11b/CD18[30, 31] or because suppressing leukocyte recruitment with blockingagents requires occupancy of >90% of active integrin receptors [2].Anti-integrin β2 antibodies have also shown unexpected side effects[33]. Additionally, whether transient activation of a fraction of nativeintegrin receptors in vivo, as is expected from treatment with anactivating agent, will have any significant biological effect inphysiologically relevant settings remains an open question.

A number of published reports in the literature show that, in additionto increasing cell adhesion and modulating migration, CD11b/CD18activation mediates a number of intracellular signaling events, mediatea number of intracellular signaling events, including production ofreactive oxygen species and modulation of a number of pro- andanti-inflammatory genes in inflammatory cells [27-32]. Integrinactivation and ligand binding leads to its clustering on the cellsurface and initiates outside-in signaling, including the activation ofPI3-K/Akt and MAPK/ERK1/2 pathways [28, 33], thereby mimicking theanchorage-dependent pro-survival signals in most cells. Ligation andclustering of integrins also synergistically potentiates intracellularsignaling by other receptors (such as, Toll-like receptors (TLRs) andcytokine receptors interleukin-1 receptor (IL-1R) and TNFR) and bothinduce transcription factor (such as, NF-κB) dependent expression ofpro-inflammatory cytokines (e.g.; IL1β, IL6, TNF-α) as well as releaseof other factors (e.g.; Tissue Factor). CD11b/CD18 deficiency enhancesTLR4-triggered production of pro-inflammatory cytokines. The abovesuggests that CD11b/CD18 and its activation has a protective role inhealthy mammals and that in inflammatory conditions or diseases,CD11b/CD18 activation would also suppress inflammation, inflammatoryinjury and disease by negatively regulating pro-inflammatory pathways inCD11b/CD18-expressing cells [34-36].

The above also suggests that there is a considerable potential foragents that modulate the function of CD11b/CD18 as therapeutic agentsfor the treatment of various inflammatory conditions. CD11b/CD18 isnormally expressed in a constitutively inactive conformation incirculating leukocytes and in many other cells, but is rapidly activatedto mediate its various biological functions [23]. CD11b/CD18 is alsoexpressed on many cell types and tissues, including microgila,hepatocytes, HSCs, HPCs and a sub-type of T- and B-cells. CD11b/CD18 isalso found in its cleaved, soluble form in some instances [37].

Blocking beta2 integrins, including CD11b/CD18, and their ligands withantibodies and ligand mimics (anti-adhesion therapy) [38-40] and geneticablation of CD11a, CD11b, CD11c or CD18 decreases the severity ofinflammatory response in vivo in many experimental models [41-43].However, such blocking agents have had little success in treatinginflammatory/autoimmune diseases in humans [42, 44], perhaps becausecomplete blockage of integrins with antibodies is difficult due toavailability of a large mobilizable intracellular pool of such integrins(for example, CD11b/CD18) [45, 46] or because suppressing leukocyterecruitment with blocking agents requires occupancy of >90% of activeintegrin receptors [47]. Anti-integrin β2 antibodies have also shownunexpected side effects [48]. Additionally, whether transient activationof a fraction of native integrin receptors in vivo, as is expected fromtreatment with an activating agent, will have any significant biologicaleffect in physiologically relevant settings remains an open question.

Therefore, there is a considerable need for novel agents, such asantibodies, proteins, peptides, chemical compounds and small molecules,that selectively regulate the ligand binding and function of β2integrins, including integrins CD11a/CD18, CD11b/CD18 and CD11c/CD18.Additionally, there is a need for agents that activate integrins(agonists). Such agonists can enhance the function of (β2 integrins by,for example, targeting or binding to an allosteric regulatory site, suchas the hydrophobic site-for-isoleucine (SILEN) pocket in CD11b/CD18, andother similar sites, but not the ligand-binding site on the integrin.Thus, there is a need for integrin activating agents that do not blockligand-binding functions of integrins. Moreover, agents and methods toenhance or promote integrin-mediated cell-adhesion and cellularfunctions are highly desired. However, progress towards identifying suchagonists has been slow, especially agonists that selectively target andactivate β2 integrins, including CD11b/CD18, with only a few reporteddiscoveries [49, 50].

The present invention describes novel CD11b/CD18 agonists and a novelapproach that involves integrin CD11b/CD18 priming for activation oractivation, rather than its blockade, as a strategy for modulatingCD11b/CD18 function. Such biological functions include cell adhesion,ligand binding, migration, phagocytosis, and the generation of effectormolecules, such as cytokines. The present invention further describescompounds and approaches for modulating the function of CD11b/CD18expressing cells (such as leukocytes, microglia, hepatocytes andlymphocytes), including their adhesion, migration, recruitment and otherbiological functions. It was strategized that various agents, such assmall molecules, which are easily delivered in vivo and can be readilyoptimized for use in different mammals, would be the best approach foractivating integrins. Here, it is shown that, without limitation,inflammatory disease can be reduced by CD11b/CD18 activation with novelsmall molecules. This shows that integrin activation is a novel, useful,pharmacologically targetable methodology to treat, without limitation, avariety of inflammatory and autoimmune diseases and conditions.

The present invention also shows that CD11b/CD18 activating agents thatactivate the normal wild type form of CD11b/CD18 and any of its mutantforms, such as the R77H mutant commonly found in many autoimmune diseasecarrying patients [51], would be highly beneficial. This inventiondescribes a novel strategy, as an alternative to the anti-adhesionstrategy that is currently practiced in literature, for regulating thebiological function of integrins and integrin-expressing cells. Manydifferent types of agents can activate integrins, such as biologics,antibodies, antibody fragments, proteins, lipids, oligonucleotides andchemical compounds.

An important requirement of useful agonists and compositions thatregulate β2 integrins, including CD11b/CD18, is that they do notnegatively impact the cell, tissue and animal viability. It is an objectof the present invention to describe such agonists, compositions andmethods. In addition, it is an objective of the present invention toshow that transient activation of a fraction of native receptors invivo, as is expected from treatment with an agonist and method of thisinvention, has a biological effect in physiologically relevant modelsystems. In addition, the present invention provides other relatedadvantages. Moreover, an important requirement of useful compounds andcompositions that regulate beta2 integrins, including CD11b/CD18, isthat they not negatively impact the cell, tissue and animal viability.Some have suggested that integrin agonists might induce killing oftarget cells (Yang et al., J Biol Chem 281, 37904 (2006)), which is notdesirable. Also, there is some prior art on the thiazolidine-one familyof compounds, including U.S. Pat. No. 5,225,426, U.S. Pat. No.7,566,732, U.S. Pat. No. 7,348,348, US 2006/0281798, US 2006/0183782, US2006/0106077, US 2008/0108677, US 2010/0056503, WO 2009026346,WO/1995/029243. However, no compounds or methods with above describeddesirable properties have so far been described in the literature. It isan object of the invention to describe such compounds and methods. Inaddition, the present invention provides other related advantages.

Furthermore, integrins are now shown to exist in more than twoconformations (closed and open). For example, CD11a/CD18 has been shownto exist in at least three conformations—closed, intermediate andopen—based on its affinity for its ligand ICAM-1 in each of these states[52]. This also suggests, although has not been previously shown, thatthese different integrin conformations will induce differentintracellular signaling pathways. It is an object of the currentinvention to describe β2 integrin agonists that, upon binding to β2integrins, activate the β2 integrins and induce intracellular signalingpathways that are different from the ligand-bound β2 integrinconformation(s).

Moreover, a number of agents currently under development asanti-inflammatory agents are targeted towards specific kinases, such asspleen tyrosine kinase (Syk), T cell receptor-associated protein kinase(ZAP70), Janus kinases (JAKs) and Bruton's tyrosine kinase (BTK) [53].There remains a need for compounds and methods that effectively treatinflammation, especially targeting those kinases.

SUMMARY OF THE INVENTION

The present invention provides for a method of treating inflammation, byadministering an effective amount of a β2 integrin agonist to a patient,and reducing inflammation.

The present invention provides for a method of treating cancer, byadministering an effective amount of a β2 integrin agonist to a patient,and reducing tumor growth.

The present invention provides for a method of treating a patientexposed to radiation, by administering an effective amount of a β2integrin agonist to the patient after radiation exposure, and mitigatingthe effects of radiation exposure in the patient.

The present invention also provides for a method of preventing effectsof radiation, by administering an effective amount of a β2 integrinagonist to the patient prior to radiation exposure, and preventing theeffects of radiation exposure on the patient.

The present invention provides for a method of treating acquired bonemarrow failure (BMF), by administering an effective amount of a β2integrin agonist to a patient.

The present invention further provides for a method of improving thehealth of damaged vasculature in a patient, by administering a β2integrin agonist to the patient, and improving re-vascularization in thepatient.

The present invention provides for a method of activating β2 integrins,by interacting the β2 integrin with an agonist, and stabilizing the b2integrin in an intermediate affinity conformation.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention are readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanying drawingswherein:

FIG. 1 is a photograph showing that β2 integrin agonist leukadherins,such as LA1, accelerate MyD88 degradation;

FIGS. 2A-2C shows that Ieukadherin LA1 reduces the rate of tumor growthupon treatment, FIG. 2A is a graph of tumor growth, FIG. 2B is an invivo bioluminescence image of a control group, and FIG. 2C is an in vivobioluminescence image of a LA1 treatment group;

FIG. 3 is a graph showing that leukadherins reduce renal injury indiabetic animals;

FIGS. 4A-4B show that increasing macrophage adhesion decreases cellmigration and in vitro wound healing, FIG. 4A is a histogram showingquantitation of the wound-healing data presented in the photograph ofFIG. 4B, representative images shown are from one well of a triplicatewell experiment from one of at least two to three independentexperiments, dotted lines represent the wound margins at the beginningof the experiment (0 hours), black scale bar represents 50 μm;

FIGS. 5A-5B show that activating antibodies, but not LA1, induceCD11b/CD18 clustering on live cells, FIG. 5A shows confocal microscopyimages of CD11b distribution on the surface of K562 WT cells, and FIG.5B is a histogram showing ImageJ based quantitation of the number ofCD11b macro clusters per cell from each of the conditions shown in FIG.5A;

FIGS. 6A-6D are graphs and immunoblots showing that activatingantibodies induce intracellular MAPK signaling that mimics ligand-boundstate, but LA1 does not, FIG. 6A is an immunoblot of whole cell lysatesfrom K562 WT cells treated in suspension for 45 min at 37° C. witheither PKC agonist PMA (positive control), DMSO (vehicle), non-selectiveagonist Mn²⁺ (1 mM), activating mAb KIM127 (1:100 dilution of ascitesfluid), activating mAb 24 (20 □g/mL) or the agonist LA1 (15 □M) probedfor phosphorylated ERK (pERK; pThr202/pTyr204) and total ERK, FIG. 6B isan immunoblot of the cell lysates probed for phosphorylated JNK (pJNK;pThr183/pTyr185) and total JNK, FIGS. 6C and 6D are immunoblots of wholecell lysates from K562 WT cells treated as in 6A and 6B but in thepresence of ligand fibrinogen (50 □g/mL);

FIGS. 7A-7B show that agonist LA1 does not induce global conformationalchanges in CD11b/CD18, FIG. 7A is a histogram showing the level ofCD11b/CD18 expression on the surface of live K562 WT cells usingheterodimer specific antibody IB4 (right) and the isotype IgG2a controlmAb (left), as measured using flow cytometry, and FIG. 7B is a FACSanalysis on live K562 WT cells showing the reactivity of CD11b/CD18conformation reporter probe antibodies KIM127 (1:50 dilution of ascites)and mAb 24 (15 ug/mL) under various conditions;

FIGS. 8A-8D show that agonists LA1 and ED7 similarly reduce the influxof macrophages in injured arteries, FIGS. 8A-8C are representative FACSanalyses plots of single cell suspensions for CD11b⁺ macrophages (basedon binding with anti-rat CD11b antibody WT.5) in arteries 7 days afterballoon injury from rats treated post-surgery with a vehicle control(8A), activating anti-CD11b mAb ED7 (3.3 mg/kg/d) (8B) or LA1 (lmg/kg/d)(8C), and FIG. 8D is a bar graph showing quantitation of CD11b⁺macrophages in the injured arteries from multiple animals (n=4-6/group);

FIGS. 9A-9D show that agonist LA1 is better at ameliorating vascularinjury as compared to activating anti-CD11b antibody ED7, FIGS. 9A-9Care representative photomicrographs of arteries 21 days after ballooninjury from rats treated post-surgery with a control irrelevant mouseIgG (mIgG1, 4 mg/kg/d) (9A), activating anti-CD11b mAb ED7 (4 mg/kg/d)(9B) or LA1 (1 mg/kg/d) (9C) (arrows point to the neoinitmalthickening), and FIG. 9D is a bar graph showing the neointima to mediaratio determined by morphometric analysis of the injured arteries fromthe treated animals (n=6-7/group);

FIG. 10A shows the chemical structure of agonist LA2, and FIG. 10B showsthe chemical structure of agonist LA15;

FIG. 11 is a histogram showing that cells expressing CD11b/CD18 showincreased adhesion in the presence of leukadherins;

FIG. 12 is a histogram showing increasing macrophage adhesion withleukadherins decreases cell migration and in vitro wound healing;

FIGS. 13A-13B show that activating antibodies, but not leukadherins LA2and LA15, induce CD11b/CD18 clustering on live cells, FIG. 13A isconfocal microscopy images of CD11b distribution on the surface of K562WT cells, FIG. 13B is a histogram showing ImageJ based quantitation ofthe number of CD11b macro clusters per cell from each of the conditionsshown in 13A above;

FIGS. 14A-14D show that agonist LA1 dose-dependently reduces vascularinjury in wild type rats, FIGS. 14A-14C are representativephotomicrographs of arteries 21 days after balloon injury from ratstreated post-surgery with vehicle control (14A), low dose of agonist LA1(0.05mg/kg/d) (14B) or a more effective dose of LA1 (1 mg/kg/d) (14C)(arrows point to the neoinitmal thickening), and FIG. 14D is a bar graphshowing the neointima to media ratio determined by morphometric analysisof the injured arteries from the treated animals (n=6-7/group).

FIGS. 15A-15G are graphs showing mRNA levels of pro-inflammatory factorsthat are upregulated by LPS treatment are significantly reduced in cellsco-treated with LA1;

FIGS. 16A-16G are graphs showing mRNA levels of pro-inflammatory factorsthat are upregulated by LPS treatment are significantly reduced in cellsco-treated with LA1;

FIG. 17 is a chart showing that levels of a number of micro RNAs aremodulated;

FIGS. 18A-18B are graphs showing cells resistant to detachment in shearflow;

FIGS. 19A-19B are graphs showing adhesive behavior of vehicle-, Mn2+ orLA-treated various CD11b/CD18 transfectants under the wall shear stressof 0.3 dyn/cm2;

FIG. 20 shows that the biochemical measurements in serum and liver ofLA1-treated rats revealed no appreciable changes in enzyme levels orserum constituents, such as proteins, cholesterol, urea and creatinine;

FIG. 21 shows no significant alterations either in relative organweights or their histology were discernible at terminal autopsy;

FIGS. 22A-22E are confocal images of DAPI-stained Human Umbilical VeinEndothelial Cells;

FIG. 23 is a graph showing leukadherin LA1 concentration in mouse bloodover time after administration via two different routes;

FIG. 24A is a graph of relative tumor growth and FIG. 24B is a chart ofrelative tumor volume;

FIG. 25 is a graph of percent survival of different treatment groups;

FIGS. 26A-26C are graphs showing the analysis of hematopoiesis and HSCcompartment in LA1 (Red bars), Vehicle control (Blue bars) treatedgroups of mice at 4 weeks after 6 Gy of total body irradiation (TBI);

FIG. 27 is a graph showing T cell proliferation with doses of LA1;

FIG. 28 is a graph of TNF-α release; and

FIG. 29 is a Western blot of Syk phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to various agents, includingchemical compounds termed leukadherins, and methods for priming oractivating β2 integrins, especially CD11b/CD18. In other words, theagents of the present invention act as agonists of β2 integrins, ratherthan antagonists. The agonists and methods are useful for treatingvarious inflammatory and autoimmune diseases, and cancer among otherdiseases.

One aspect of the invention relates to a compound of Formula (I)

wherein

A is absent or is selected from alkyl and alkenyl;

B is absent or is selected from alkyl, alkenyl, O, S and NR⁴;

N is nitrogen;

X and Y are independently selected from O and S;

Z is selected from CR⁴, O, S and NR⁴;

U, V and W are independently selected from CR⁴, O, S and NR⁴;

R¹ and R³ are independently selected from acyl, alkyl, alkenyl, alkynyl,hydroxyalkyl, aminoalkyl, thioalkyl, aryl, aralkyl, carboxyaryl,alkoxyalkyl, alkoxyaryl, alcoxycarbonylaryl, aminoaryl, amidoaryl,haloaryl, heteroaryl, heteroaralkyl, carbocyclyl, heterocyclyl,heterocyclylalkyl, alkoxycarbonyl, alkylaminocarbonyl,alkylthiocarbonyl, sulfonate, alkylsulfonate, arylsulfonate, sulfone,alkylsulfone, arylsulfone, sulfoxide, alkylsulfoxide, arylsulfoxide,alkylsulfonamide, arylsulfonamide, and sulfonamide, piperidinyl,morpholinyl, pyrrolidinyl, phenyl, pyridyl, pyrimidinyl, furyl, thienyl,pyrrolyl, imidazolyl, triazolyl, pyrazolyl, thiazolyl, isoxazolyl,oxazolyl, oxadiazolyl, indolyl, naphthyl, quinolinyl, isoquinolinyl,quinoxalinyl, benzyl, benzofuryl, dibenzofuryl, benzthienyl,benzoxazolyl, benzothiazolyl, benzimidazolyl, pyridoimidazolyl,pyrimidoimidazolyl, pyridopyrazolyl, pyrazolopyrimidinyl, and any ofwhich is optionally substituted with 1-6 independent substituents;

R² selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl, thioalkyl,alkoxyalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl; and

R⁴ is absent or is selected from hydrogen and alkyl.

In certain embodiments, Z is S. In certain embodiments, X and Y are O.In certain other embodiments, X and Y are S. In certain otherembodiments, X is S and Y is O. In certain other embodiments, X and Yare O and Z is S. In certain other embodiments, X, Y and Z are S. Incertain other embodiments, X and Z are S and Y is O. In certainembodiments, U is O and V and W are CR⁴. In certain such embodiments, R⁴is hydrogen. In certain other embodiments, U is S and V and W are CR⁴.In certain such embodiments, R⁴ is hydrogen. In certain otherembodiments, U is CR⁴, V is N and W is O. In certain such embodiments,R⁴ is hydrogen. In certain other embodiments, U is CR⁴, V is O and W isN. In certain such embodiments, R⁴ is hydrogen. In certain embodiments,B is alkyl and A is absent. In certain such embodiments, R¹ is selectedfrom alkoxycarbonyl, aryl, heteroaryl, carbocyclyl, heterocyclyl andalkoxycarbonyl. In certain embodiments, B is methylene and A is absent.In certain such embodiments, R¹ is selected from alkoxycarbonyl, aryl,heteroaryl, carbocyclyl, heterocyclyl and alkoxycarbonyl. In certainsuch embodiments, B is methylene and A is absent. In certainembodiments, where A is alkyl and B is absent, R¹ is alkoxycarbonyl. Incertain embodiments, A and B are both absent. In certain suchembodiments, R¹ is selected from alkoxycarbonyl, aryl, heteroaryl,carbocyclyl, heterocyclyl and alkoxycarbonyl. In certain embodiments, R¹substituent is further substituted with 1-6 independent substituents. Incertain embodiments, R¹ is selected from furan, phenyl, benzyl,tetrahydrofuran, tetrahydrothiophene, pyrrolidine, tetrahydropyran,tetrahydrothiopyran, piperidine, piperazine, and morpholine. In certainembodiments R¹ is selected from tetrahydrofuran, tetrahydrothiophene,and pyrrolidine, preferably tetrahydrofuran. In certain embodiments, R¹is phenyl, preferably substituted phenyl. In certain such embodiments,R¹ is phenyl substituted one to five, preferably one to three, morepreferably one or two times. In certain such embodiments, R¹ is phenylsubstituted with one or two, preferably one substituent independentlyselected from halogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy,alkylamino, alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl,and alkyl, more preferably from alkyl and halogen, e.g., from methyl,fluoro and chloro. In certain embodiments, R² is selected from hydrogen,alkyl, hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, R³ is selected fromalkyl, hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, R² is hydrogen and R³ isselected from aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl, preferably aryl,heteroaryl, carbocyclyl, and heterocyclyl. In certain embodiments, R³ isheteroaryl selected from pyrrole, furan, pyrimidine, oxazole, isooxazoleand thiophene, preferably furan. In certain embodiments, R³ is furansubstituted one to three, preferably one to two times, more preferablyonce. In certain such embodiments, R³ is furan substituted once with asubstituent selected from aryl, aralkyl, heteroaryl, heteroaralkyl,carbocyclyl, carbocyclylalkyl, heterocyclyl and heterocyclylalkyl,preferably aryl, heteroaryl, carbocyclyl, and heterocyclyl. In certainembodiments, R³ is furan substituted once with an aryl group, whichitself is optionally substituted, preferably one to two times withalkyl, carboxyl, alkoxycarbonyl and halogen, e.g., chlorophenyl,dichlorophenyl, carboxyphenyl. In certain embodiments, R³ is aryl,preferably phenyl. In certain such embodiments, R³ is phenyl substitutedwith one or two, preferably two substituents independently selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl.In certain such embodiments, R³ is phenyl substituted once with ahalogen, preferably bromo.

One aspect of the invention relates to a compound of Formula (II)

wherein

A is absent or is selected from alkyl and alkenyl;

B is absent or is selected from alkyl, alkenyl, O, S and NR⁴;

N is nitrogen;

X and Y are independently selected from O and S;

Z is selected from CR⁴, O, S and NR⁴;

U, V and W are independently selected from CR⁴, O, S and NR⁴;

R¹ and R³ are independently selected from acyl, alkyl, alkenyl, alkynyl,hydroxyalkyl, aminoalkyl, thioalkyl, aryl, aralkyl, carboxyaryl,alkoxyalkyl, alkoxyaryl, alcoxycarbonylaryl, aminoaryl, amidoaryl,haloaryl, heteroaryl, heteroaralkyl, carbocyclyl, heterocyclyl,heterocyclylalkyl, alkoxycarbonyl, alkylaminocarbonyl,alkylthiocarbonyl, sulfonate, alkylsulfonate, arylsulfonate, sulfone,alkylsulfone, arylsulfone, sulfoxide, alkylsulfoxide, arylsulfoxide,alkylsulfonamide, arylsulfonamide, and sulfonamide, piperidinyl,morpholinyl, pyrrolidinyl, phenyl, pyridyl, pyrimidinyl, furyl, thienyl,pyrrolyl, imidazolyl, triazolyl, pyrazolyl, thiazolyl, isoxazolyl,oxazolyl, oxadiazolyl, indolyl, naphthyl, quinolinyl, isoquinolinyl,quinoxalinyl, benzyl, benzofuryl, dibenzofuryl, benzthienyl,benzoxazolyl, benzothiazolyl, benzimidazolyl, pyridoimidazolyl,pyrimidoimidazolyl, pyridopyrazolyl, pyrazolopyrimidinyl, and any ofwhich is optionally substituted with 1-6 independent substituents;

R² selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl, thioalkyl,alkoxyalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl; and

R⁴ is absent or is selected from hydrogen and alkyl.

In certain embodiments, Z is S. In certain embodiments, X and Y are O.In certain other embodiments, X and Y are S. In certain otherembodiments, X is S and Y is O. In certain other embodiments, X and Yare O and Z is S. In certain other embodiments, X, Y and Z are S. Incertain other embodiments, X and Z are S and Y is O. In certainembodiments, U is O and V and W are CR⁴. In certain such embodiments, R⁴is hydrogen. In certain other embodiments, U is S and V and W are CR⁴.In certain such embodiments, R⁴ is hydrogen. In certain otherembodiments, U is CR⁴, V is N and W is O. In certain such embodiments,R⁴ is hydrogen. In certain other embodiments, U is CR⁴, V is O and W isN. In certain such embodiments, R⁴ is hydrogen. In certain embodiments,B is alkyl and A is absent. In certain such embodiments, R¹ is selectedfrom alkoxycarbonyl, aryl, heteroaryl, carbocyclyl, heterocyclyl andalkoxycarbonyl. In certain embodiments, B is methylene and A is absent.In certain such embodiments, R¹ is selected from alkoxycarbonyl, aryl,heteroaryl, carbocyclyl, heterocyclyl and alkoxycarbonyl. In certainsuch embodiments, B is methylene and A is absent. In certainembodiments, where A is alkyl and B is absent, R¹ is alkoxycarbonyl. Incertain embodiments, A and B are both absent. In certain suchembodiments, R¹ is selected from alkoxycarbonyl, aryl, heteroaryl,carbocyclyl, heterocyclyl and alkoxycarbonyl. In certain embodiments, R¹substituent is further substituted with 1-6 independent substituents. Incertain embodiments, R¹ is selected from furan, phenyl, benzyl,tetrahydrofuran, tetrahydrothiophene, pyrrolidine, tetrahydropyran,tetrahydrothiopyran, piperidine, piperazine, and morpholine. In certainembodiments R¹ is selected from tetrahydrofuran, tetrahydrothiophene,and pyrrolidine, preferably tetrahydrofuran, In certain embodiments, R¹is phenyl, preferably substituted phenyl. In certain such embodiments,R¹ is phenyl substituted one to five, preferably one to three, morepreferably one or two times. In certain such embodiments, R¹ is phenylsubstituted with one or two, preferably one substituent independentlyselected from halogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy,alkylamino, alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl,and alkyl, more preferably from alkyl and halogen, e.g., from methyl,fluoro and chloro. In certain embodiments, R² is selected from hydrogen,alkyl, hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, R³ is selected fromalkyl, hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, R² is hydrogen and R³ isselected from aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl, preferably aryl,heteroaryl, carbocyclyl, and heterocyclyl. In certain embodiments, R³ isheteroaryl selected from pyrrole, furan, pyrimidine, oxazole, isooxazoleand thiophene, preferably furan. In certain embodiments, R³ is furansubstituted one to three, preferably one to two times, more preferablyonce. In certain such embodiments, R³ is furan substituted once with asubstituent selected from aryl, aralkyl, heteroaryl, heteroaralkyl,carbocyclyl, carbocyclylalkyl, heterocyclyl and heterocyclylalkyl,preferably aryl, heteroaryl, carbocyclyl, and heterocyclyl. In certainembodiments, R³ is furan substituted once with an aryl group, whichitself is optionally substituted, preferably one to two times withalkyl, carboxyl, alkoxycarbonyl and halogen, e.g., chlorophenyl,dichlorophenyl, carboxyphenyl. In certain embodiments, R³ is aryl,preferably phenyl. In certain such embodiments, R³ is phenyl substitutedwith one or two, preferably two substituents independently selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl.In certain such embodiments, R³ is phenyl substituted once with ahalogen, preferably bromo.

In certain embodiments, a compound of Formula II is selected from

In certain embodiments, a compound of Formula II selected from thefollowing compounds is less preferred

One aspect of the invention relates to a compound of Formula (Ill)

wherein

A is absent or is selected from alkyl and alkenyl;

B is absent or is selected from alkyl, alkenyl, O, S and NR⁴;

N is nitrogen;

X and Y are independently selected from O and S;

Z is selected from CR⁴, O, S and NR⁴;

U, V and W are independently selected from CR⁴, O, S and NR⁴;

R³ is 1-6 independent substituents present at position(s) 1-6 of thearyl ring;

R¹ and R³ are independently selected from acyl, alkyl, alkenyl, alkynyl,hydroxyalkyl, aminoalkyl, thioalkyl, aryl, aralkyl, carboxyaryl,alkoxyalkyl, alkoxyaryl, alcoxycarbonylaryl, aminoaryl, amidoaryl,haloaryl, heteroaryl, heteroaralkyl, carbocyclyl, heterocyclyl,heterocyclylalkyl, alkoxycarbonyl, alkylaminocarbonyl,alkylthiocarbonyl, sulfonate, alkylsulfonate, arylsulfonate, sulfone,alkylsulfone, arylsulfone, sulfoxide, alkylsulfoxide, arylsulfoxide,alkylsulfonamide, arylsulfonamide, and sulfonamide, piperidinyl,morpholinyl, pyrrolidinyl, phenyl, pyridyl, pyrimidinyl, furyl, thienyl,pyrrolyl, imidazolyl, triazolyl, pyrazolyl, thiazolyl, isoxazolyl,oxazolyl, oxadiazolyl, indolyl, naphthyl, quinolinyl, isoquinolinyl,quinoxalinyl, benzyl, benzofuryl, dibenzofuryl, benzthienyl,benzoxazolyl, benzothiazolyl, benzimidazolyl, pyridoimidazolyl,pyrimidoimidazolyl, pyridopyrazolyl, pyrazolopyrimidinyl, and any ofwhich is optionally substituted with 1-6 independent substituents;

R² selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl, thioalkyl,alkoxyalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl; and

R⁴ is absent or is selected from hydrogen and alkyl.

In certain embodiments, Z is S. In certain embodiments, X and Y are O.In certain other embodiments, X and Y are S. In certain otherembodiments, X is S and Y is O. In certain other embodiments, X and Yare O and Z is S. In certain other embodiments, X, Y and Z are S. Incertain other embodiments, X and Z are S and Y is O. In certainembodiments, U is N and V and W are CR⁴. In certain such embodiments, R⁴is hydrogen. In certain other embodiments, V is N and U and W are CR⁴.In certain such embodiments, R⁴ is hydrogen. In certain otherembodiments, W is N and V and V are CR⁴. In certain such embodiments, R⁴is hydrogen. In certain embodiments, B is alkyl and A is absent. Incertain such embodiments, R¹ is selected from alkoxycarbonyl, aryl,heteroaryl, carbocyclyl, heterocyclyl and alkoxycarbonyl. In certainembodiments, B is methylene and A is absent. In certain suchembodiments, R¹ is selected from alkoxycarbonyl, aryl, heteroaryl,carbocyclyl, heterocyclyl and alkoxycarbonyl. In certain suchembodiments, B is methylene and A is absent. In certain embodiments,where A is alkyl and B is absent, R¹ is alkoxycarbonyl. In certainembodiments, A and B are both absent. In certain such embodiments, R¹ isselected from alkoxycarbonyl, aryl, heteroaryl, carbocyclyl,heterocyclyl and alkoxycarbonyl. In certain embodiments, R¹ substituentis further substituted with 1-6 independent substituents. In certainembodiments, R¹ is selected from furan, phenyl, benzyl, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, tetrahydropyran, tetrahydrothiopyran,piperidine, piperazine, and morpholine. In certain embodiments R¹ isselected from tetrahydrofuran, tetrahydrothiophene, and pyrrolidine,preferably tetrahydrofuran. In certain embodiments, R¹ is phenyl,preferably substituted phenyl. In certain such embodiments, R¹ is phenylsubstituted one to five, preferably one to three, more preferably one ortwo times. In certain such embodiments, R¹ is phenyl substituted withone or two, preferably one substituent independently selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl,more preferably from alkyl and halogen, e.g., from methyl, fluoro andchloro. In certain embodiments, R² is selected from hydrogen, alkyl,hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, R³ is selected fromalkyl, hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, R² is hydrogen and R³ isselected from aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl, preferably aryl,heteroaryl, carbocyclyl, and heterocyclyl. In certain embodiments, R³ isheteroaryl selected from pyrrole, furan, pyrimidine, oxazole, isooxazoleand thiophene, preferably furan. In certain embodiments, R³ is furansubstituted one to three, preferably one to two times, more preferablyonce. In certain such embodiments, R³ is furan substituted once with asubstituent selected from aryl, aralkyl, heteroaryl, heteroaralkyl,carbocyclyl, carbocyclylalkyl, heterocyclyl and heterocyclylalkyl,preferably aryl, heteroaryl, carbocyclyl, and heterocyclyl. In certainembodiments, R³ is furan substituted once with an aryl group, whichitself is optionally substituted, preferably one to two times withalkyl, carboxyl, alkoxycarbonyl and halogen, e.g., chlorophenyl,dichlorophenyl, carboxyphenyl. In certain embodiments, R³ is aryl,preferably phenyl. In certain such embodiments, R³ is phenyl substitutedwith one or two, preferably two substituents independently selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl.In certain such embodiments, R³ is phenyl substituted once with ahalogen, preferably bromo.

The compounds of the present invention can have an inherent end-to-endpolarity such that compounds are more polar on one end of the molecule,for example on the top-end (N-substituted side of the thiazolidine ring)or the bottom-end (substituted furanyl side of the thiazolidine ring) asdrawn, as compared to the other end of the molecule. Alternatively,compounds with two polar ends can be disfavored.

The compounds of the invention can be in a pure or substantially puresingle configuration, such as a Z-configuration.

Certain compounds of the present invention can exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (d)-isomers, (I)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms can bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

One aspect of the invention relates to non-blocking ligands of β2integrins. In one aspect, the β2 (b2) integrin is CD11b/CD18.Non-limiting examples of non-blocking ligands is CD40L, soluble CD40L(sCD40L), uPAR and soluble uPAR (suPAR). In one aspect, the non-blockingligands of b2 integrins are integrin agonists, such that they activateb2 integrins. In a related aspect, the non-blocking ligands of b2integrins activate b2 integrins and promote their binding to otherligands of b2 integrins. As an example, sCD40L can be used to promoteactivation of CD11b/CD18 and binding of CD11b/CD18 to fibrinogen,ICAM-1, uPAR and iC3b.

One aspect of the invention relates to compounds and methods thatregulate binding of non-blocking ligands of β2 integrins to β2integrins. In one aspect, the β2 (b2) integrin is CD11b/CD18.Non-limiting examples of non-blocking ligands is CD40L, soluble CD40L(sCD40L), uPAR and soluble uPAR (suPAR). In one aspect, the compounds ofthe present invention regulate the binding of the non-blocking ligandsof CD11b/CD18, such as sCD40L and suPAR, to CD11b/CD18. In a relatedaspect, the regulation of binding of the non-blocking ligands of b2integrins to b2 integrins by the compounds of this invention changesintracellular signaling in the b2 integrin expressing cells.

The present invention provides for a method of treating inflammation, byadministering an effective amount of a β2 integrin agonist to a patient,and reducing inflammation. Preferably, the β2 integrin is CD11b/CD18.Inflammation can be reduced by reducing inflammatory cell migration andrecruitment by increasing CD11b/CD18-mediated cell adhesion. Theinflammatory cells can be macrophages, or any other cells thatcontribute to inflammation.

Inflammation can also be reduced by binding the (β2 integrin agonist toan allosteric pocket in the αA-domain in CD11b. Such binding occurswithout inducing global conformational changes in CD11b/CD18 andprevents outside-in signaling. The binding further induces priming ofCD11b/CD18 and converts CD11b/CD18 into a stabilized intermediateconformation. The binding of the β2 integrin agonist preferably occurswith one or more residues within the E162-L170 sequence of CD11b, i.e.SEQ ID NO: 1 (EQLKKSKTL).

Treating inflammation can further include reducing mechanical vascularinjury and preventing and reducing neointimal hyperplasia. Inflammationcan also be reduced by accelerating degradation of MyD88, inducingfaster dampening of TLR4-mediated pathways, and inducing Sykphosphorylation. Various pro-inflammatory factors can be upregulated ordownregulated by the administration of the β2 integrin agonist. Factorsthat can be upregulated include hsa-miR-125b, hsa-miR-330-3p,hsa-miR-363, hsa-miR-134, hsa-miR-523, hsa-miR-1266, hsa-miR-15b*,hsa-miR-877*, hsa-miR-130b*, hsa-miR-1237, hsa-miR-26b*, hsa-miR-191*,and combinations thereof. Factors that can be downregulated includehsa-miR-181d, hsa-miR-151-3p, hsa-miR-526b, hsa-miR-199α-3p,hsa-miR-361-5p, hsa-miR-95, hsa-miR-551a, hsa-miR-365*, hsa-miR-1908,hsa-miR-624*, hsa-miR-1913, hsa-miR-330-5p, hsa-miR-520d-3p,hsa-miR-224*, hsa-miR-505*, and combinations thereof.

The present invention also provides for a method of treating cancer, byadministering an effective amount of a β2 integrin agonist to a patient,and reducing tumor growth. Reducing tumor growth can involve reducingincidence and size of metastases, reducing the rate of tumor regrowth,reducing the amount of inflammatory leukocytes, reducing tumorvascularization, reducing tumor engraftment, and combinations thereof.Reducing tumor growth can further involve reducing T-cell proliferation,decreasing IFN-g production by T-cells, reducing TNF-α release.

One aspect of the invention relates to compositions and methods ofinducing an intermediate form of b2 integrins by binding of an integrinagonist to a b2 integrin. In particular, the present invention isdirected towards CD11b/CD18, where binding of novel agonists of thisinvention induces an intermediate conformation of CD11b/CD18 onCD11b/CD18 expressing cells. Induction of the intermediate conformationleads to different type of intracellular signaling than the signalingthat is induced upon integrin activation with protein ligands. Thebinding of an agonist of the invention to a b2 integrin can inducedifferent intracellular signaling than binding of an activating antibodyto a b2 integrin. Therefore, the present invention generally providesfor a method of activating β2 integrins, by interacting the β2 integrinwith an agonist, and stabilizing the b2 integrin in an intermediateaffinity conformation.

The agonists and methods of the present invention can be used incombination with agents targeting Syk, Btk, JAK, JAK1, JAK2, JAK3 andtheir dosing can be increased or can be lowered in combination with thecompounds of the present invention. Ant-clotting drugs, steroids,sphingosine-phosphate receptor modulators, and drug-eluting stent mediacan also be administered. Due to the synergy with any of thesecompounds, doses can be lowered and negative side effects can be reducedor eliminated associated with typical doses.

The agonists and methods of the present invention can be used incombination with anti-inflammatory drugs, such as, but not limited to,non-steroidal anti-inflammatory drugs (NSAIDS) such as salicylates(aspirin), acetic acid derivatives (indomethacin), propionic acidderivatives (ibuprofen or naproxen), or CoxII inhibitors such ascelecoxib (CELEBREX®, Pfizer, Inc.) or rofecoxib (VIOXX®, Merck). Dosingis generally 10 to 3200 mg for anti-inflammatory drugs per day, whichcan be lowered in combination with the agonists of the present inventiondue to synergy of the combination. Furthermore, due to the synergy, sideeffects can be reduced that are associated with a typical dose of theanti-inflammatory drugs.

The agonists and methods of the present invention can be used incombination with anti-cancer compounds such as, but not limited to,cilengitide, a cyclo(RGDfV) peptide. Dosing can generally be 120 to 2400mg/m², and can be lowered in combination with the compounds of thepresent invention due to synergy of the combination, therefore reducingside effects associated with a typical dose of the anti-cancercompounds.

The agonists and methods of the present invention can be used incombination with anti-rejection drugs, such as, but not limited to,tacrolimus, cyclosporine, and various steroids. Dosing for tacrolimuscan be 0.25 mg to 1 mg per day and can be lowered in combination withthe agonists of the present invention. Dosing for cyclosporine can be 1to 12 mg/kg per day and can be lowered in combination with the agonistsof the present invention due to synergy of the combination.

The agonists and methods of the present invention can be used incombination with anti-cancer treatments, such as chemotherapy,radiation, or surgery. Dosing of the anti-cancer treatments can belowered in combination with the compounds of the present invention dueto synergy of the combination, and therefore reducing side effectsassociated with typical dosing of these treatments. Compounds of thepresent invention can also be used as adjuvants to various anti-cancertreatments, such as chemotherapy, radiation and surgery. Compounds ofthe present invention reduce the growth of tumors, re-growth of tumors,tumor vascularization, recruitment of leukocytes in response to tumorcells or injury to tumors, tumor metastasis, tumor engraftment, andobesity and its response to tumors.

The agonists of the present invention can also be used to preventradiation exposure induced injury in patients and cells. The presentinvention provides for a method of preventing effects of radiation, byadministering an effective amount of a β2 integrin agonist to a patientprior to radiation exposure, and preventing the effects of radiationexposure on the patient.

The compounds of the present invention can also be used to mitigate theeffects of radiation exposure. In particular, the compounds of thepresent invention can be administered to patients after the radiationexposure (delayed administration). The compounds of the presentinvention also protect various organs and compartments from radiationdamage, including the hematopoietic system. Compounds of the presentinvention are effective radiomitigants and can be used under a delayedtreatment scenario. Additionally, treatment with compounds of thepresent invention accelerates recovery of hematopoiesis and results insignificant improvement in the recovery of HSC compartment afterradiation exposure, including sublethal radiation. Therefore, thepresent invention provides for a method of treating a patient exposed toradiation, by administering an effective amount of a β2 integrin agonistto the patient after radiation exposure, and mitigating the effects ofradiation exposure in the patient.

Acquired bone marrow failure (BMF) develops after an injury to the bonemarrow (BM) by ionizing radiation (IR), chemotherapy drugs andantibiotics (e.g. busulfan and chloramphenicol), toxic chemicals(benzene, carbon tetrachloride), or viral infection (hepatitis, HIV,CMV, parvovirus) (1). Another form of acquired BMF called aplasticanemia is an immune-mediated BMF that develops after lymphocyteinfusion, and is characterized by an immune-mediated functionalimpairment of hematopoietic stem cells (HSCs). The agonists of thepresent invention can also be used to prevent, mitigate or delay thedevelopment acquired BMF or reduce the amplitude of acquired BMF.Therefore, the present invention provides for a method of treatingacquired bone marrow failure (BMF), by administering an effective amountof a β2 integrin agonist to a patient.

The agonists of the present invention can further be used for improvingre-vascularization in patients with vascular wall damage. The presentinvention also provides for a method of improving the health of damagedvasculature in a patient by administering a β2 integrin agonist to thepatient, and improving re-vascularization in the patient.

The present invention provides for a method of activating β2 integrinsby interacting the β2 integrin with an agonist, preferably one of thecompounds described herein. The compounds of the present invention canstabilize the b2 integrin in an intermediate affinity conformation.

The agonists can also correct or reduce the functional deficit in cellsthat express mutant forms of β2 integrins. For example, mutations inCD11b, such as the R77H mutation, have been linked to lupus and lupusnephritis. The agonists of the present invention can reduce or overcomethe functional defects in cells, organisms, and animals that carrymutant forms of the β2 integrins. Therefore, diseases or conditions canbe treated or prevented that are associated with the activity of β2integrins, such as, diabetic nephropathy, lupus and lupus nephritis.

More specifically, the present invention provides for a method oftreating nephropathy, by administering an effective amount of a β2integrin agonist to a patient, and improving the health of the patient'skidneys. The nephropathy can be diabetic nephropathy. The health of thekidneys is improved by reducing the number of infiltrating leukocytes inthe kidneys, preserving kidney function, and reducing glomerular damageand glomerular mesangial sclerosis.

The agonists of the present invention can also more generally modulatebiological function in vitro or in vivo, such as, but not limited to,gene expression, epigenetic profile, protein expression, protein levels,protein modifications, post-translational modifications, and signaling.Preferably, the agonists of the invention modulate biological functionin leukocytes, microglia and stem cells. Alternatively, the agonists ofthe invention can modulate biological function in other cells ortissues.

The agonists of the present invention can also modulate other biologicalfunctions in vitro or in vivo, such as, differentiation of stem cells,differentiation of pluripotent cells, maintenance of cells in culture orin long term storage, mobilization of cells, such as leukocytes frombone marrow into circulation or endothelial progenitor cells to sites ofinflammation or injury and increasing retention of certain cells intotheir niches, such as leukemia cells in the marrow.

The treatment of the patient in any of the above methods can beconfirmed by detecting the activation of the (β2 integrins. This can beaccomplished by taking a sample from the patient and performing anassay, such as detection of levels of β2 integrin expression on thesurface of leukocytes in the biological sample or the level of activatedβ2 integrin on such cells. Another approach for confirming the treatmentof a patient is to evaluate levels of the other known markers in thepatient that are typically associated with the said disease, such aslevels of IL-6 in the blood samples, or disease symptoms in the patient.

Computer-based modeling algorithms can be used to analyze the structuresand conformations of agonists that bind β2 integrins, especiallyCD11b/CD18, to identify structural features that contribute tosuccessful binding. Such information can be analyzed in conjunction withinformation about the structure or conformation of CD11b/CD18 or abinding pocket thereof, such as structural information obtained byanalysis of CD11b/CD18 using analytical techniques such as x-raycrystallography or nuclear magnetic resonance, to analyze interactionsbetween binding agonists and the binding pocket they interact with. Suchanalysis can be used to predict the portion of CD11b/CD18 that interactswith the agonist, to select agonists that possess structural featurescorrelated with desired binding activity from a library of testagonists, or to design structures that are expected to exhibit bindingwith CD11b/CD18 for testing in vivo or in vitro using assays asdescribed herein.

The computer-based modeling algorithms can also be used to identifynovel agonists that bind β2 integrins, especially CD11b/CD18, usingstructural features of the chemical compound agonists of this invention.Scaffold hopping, atom replacement, residue replacement and/or moleculereplacement methods can be used. The information can be analyzed inconjunction with information about the structure or conformation ofCD11b/CD18 or a binding pocket thereof, such as structural informationobtained by analysis of CD11b/CD18 using analytical techniques such asx-ray crystallography or nuclear magnetic resonance, to analyzeinteractions between binding agonists and the binding pocket theyinteract with. Such analysis can be used to predict the portion ofCD11b/CD18 that interacts with the agonist, to select agonists thatpossess structural features correlated with desired binding activityfrom a library of test agonists, or to design structures that areexpected to exhibit binding with CD11b/CD18 for testing in vivo or invitro using assays as described herein.

A method of detecting or diagnosing a condition or disease in a patientis provided, by administering a β2 integrin agonist as described herein,detecting binding of the β2 integrin agonist to a β2 integrin, andconfirming the presence of the disease. Preferably, the β2 integrin isCD11b/CD18. In other words, if binding is present, the patient has adisease as described above. For example, the disease can be aninflammatory disease or autoimmune disease, and by detecting the bindingof the agonist to CD11b/CD18, it can be confirmed that a patient hasthose diseases. Also an agonist of the present invention can beadministered to biological samples obtained from a patient in order todetect or diagnose a condition or a disease in a patient. Theadministered agonist can be derivatized, tagged, polymerized,encapsulated or embedded in such a way that it allows easy detection.The agonist can be tagged with a tracer, a radio-label or a fluorescenttag using a linker. The agonist can be detected using Magnetic ResonanceImaging (MRI) and other such diagnostic techniques as known in the art.Another method of detection can be as follows. A biological sample canbe taken from a patient, such as blood or plasma, and an assay can beperformed, such as to detect the binding of the β2 integrin agonist tothe β2 integrin or measuring other markers (for example, IL-6 levels) inthe sample.

The present invention also provides for a method of improving thegeneral wellness of a patient by administering an effective amount of aβ2 integrin agonist, and activating β2 integrins. In other words, byadministering the agonists of the present invention, a patient's healthand wellness improves because the agonists treat many different diseasesas described above.

The dosage of the agonists and/or compositions of the invention can varydepending on many factors such as the pharmacodynamic properties of theagonist, the mode of administration, the age, health and weight of therecipient, the nature and extent of the symptoms, the frequency of thetreatment and the type of concurrent treatment, if any, and theclearance rate of the agonist in the animal to be treated. One of skillin the art can determine the appropriate dosage based on the abovefactors. The agonists of the invention can be administered initially ina suitable dosage that can be adjusted as required, depending on theclinical response.

The newly described small molecule agonists (termed “leukadherins”)activate CD11b/CD18 by binding to the integrin's ligand-binding αAdomain (also known as the CD11bA-domain and the aI-domain [54]), anapproximately 200 amino acid von Willebrand factor type A (VWFA) domainin the CD11b chain. Modeling studies showed that leukadherins bind to anallosteric pocket in the αA domain, shifting the equilibrium to its moreactive conformation, thereby promoting ligand engagement by CD11b/CD18[55]. Leukadherin-mediated integrin activation increasesCD11b/CD18-dependent adhesion of leukocytes, which leads to asignificant reduction in their migration in vitro and in vivo andresults in a significant decrease in inflammatory injury. A number ofmonoclonal antibodies (mAbs) that activate CD11b/CD18 and other b2integrins or that bind in an activation-sensitive manner (togetherreferred to as “activating mAbs”) have also been previously described inthe literature [56-65]. KIM127 is an activation-dependent antibody thatalso activates human CD11b/CD18 by recognizing sites in the CD18 EGF2domain that are buried in the inactive integrin conformation [57, 61,66]. Antibody 24 (mAb 24) detects and stabilizes the ligand-bound activeconformation of human b2 integrins and recognizes anactivation-sensitive epitope in the CD18 A-domain (αA domain) [59].Similarly, activating antibodies against murine and rat b2 integrinshave also been described in the literature. M18/2 recognizes the murineCD18 chain and simulates CD11b/CD18-dependent cell adhesion androsetting [67-69]. The anti-rat CD11b antibodies ED7 and ED8 enhanceCD11b/CD18-dependent granulocyte adhesion and homotypic aggregation,suggesting that they activate CD11b/CD18 [70].

As a therapeutic agent, the small molecule compounds and theantibody-based biologics each have distinct advantages anddisadvantages. While small molecules are easily delivered (typicallyorally), they are rapidly cleared and require frequent dosing, althoughthe oral route of administration makes it an easy process. The route ofadministration of antibody-based biological agents is less thandesirable, as they are typically injected intravenously into thecirculation, although their long in vivo half-life means that they needto be typically administered weekly or every other week. However, thisdelayed clearance of antibody-based biologics is also a liability, incase they lead to serious side effects, as the side effects take a muchlonger time to subside. Additionally, biologics have the potential todevelop an immune response against them, generating new complications inthe treated patients. Having established that CD11b/CD18 activation is anovel and pharmacologically useful mechanism for the development ofanti-inflammatory therapeutics, we wondered if both types of integrinagonists—small molecule based chemical compounds and the antibody basedbiologics—would be equally effective and reasonable to use in vivo totreat inflammation via this mechanism of action (MOA). To address thisquestion, a head-to-head testing was performed of the two types ofagents using the newly developed leukadherins compounds and a number ofanti-CD11b/CD18 activating antibodies that are widely available.

Here, the findings are reported that indeed CD11b/CD18 activation viaboth types of reagents (the chemical leukadherins and the biologicactivating mAbs) increases integrin-mediated cell adhesion and decreasescell migration and wound healing in vitro, showing that both types ofagents have a similar mechanism of action. However, it is also shownthat while leukadherins do not induce CD11b/CD18 clustering on cellsurface or intracellular signaling pathways in the treated cells, theactivating antibodies produced significant CD11b/CD18 clustering andincreased phosphorylation of key intracellular signaling proteins. Thisshows that unlike the binding with leukadherins, engagement ofCD11b/CD18 with activating mAbs mimics a ligand-bound state and inducessignificant outside-in signaling. Further mechanistic investigationsusing conformation-specific probes showed that leukadherin binding didnot induce large global conformational changes in CD11b/CD18 that aretypically associated with binding of ligands or activating antibodies[28, 71, 72]. This explains the lack of ligand-mimetic outside-insignaling by leukadherins. Finally, in a head-to-head comparison in avascular injury model in vivo, while both LA1 and ED7 similarly andsignificantly reduced the influx of macrophages into the injuredarteries, only leukadherin LA1 dose-dependently reduced vascular injuryand showed significantly higher efficacy than the anti-CD11b activatingantibody ED7. The results show that these small molecule agonists ofCD11b/CD18 have a clear therapeutic advantage over the biologicactivating antibody. Together, the data presented here show that smallmolecule agonists of CD11b/CD18 have significant advantages overbiological agonists, which can require significant optimization beforebiologic agonists can be used in vivo to take advantage of this newmechanism of action for the development of novel anti-inflammatorytherapeutics. Thus, leukadherins represent a preferred class of agentsfor development into future anti-inflammatory therapeutics.

The compound of the present invention is administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compound of the presentinvention can be administered in various ways. It should be noted thatit can be administered as the compound and can be administered alone oras an active ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The compounds can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally,intratonsillar, and intranasal administration as well as intrathecal andinfusion techniques. Implants of the compounds are also useful. Thepatient being treated is a warm-blooded animal and, in particular,mammals including man. The pharmaceutically acceptable carriers,diluents, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

The doses can be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include; U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

EXAMPLE 1

Methods of Synthesis.

Compounds of the present invention may be readily synthesized usingtechniques known to those skilled in the art, such described, forexample, in Advanced Organic Chemistry. March, 4th Ed., John Wiley andSons, New York, N.Y., 1992; Advanced Organic Chemistry, Carey andSundberg, Vol. A and B, 3rd Ed., Plenum Press, Inc., New York, N.Y.,1990; Protective groups in Organic Synthesis, Green and Wuts, 2″d Ed.,John Wiley and Sons, New York, N.Y., 1991; Comprehensive OrganicTransformations, Larock, VCH Publishers, Inc., New York, N.Y., 1988 andreferences cited therein. The starting materials for the compoundsdescribed in this invention may be prepared using standard synthetictransformations of chemical precursors that are readily available fromcommercial sources, such as, Aldrich Chemical Co. (Milwaukee, Wis.);Sigma Chemical Co. (St. Louis, Mo.); Lancaster Synthesis (Windham,N.H.); Ryan Scientific (Columbia, S.C.); Maybridge (Cornwall, UK);Matrix Scientific (Columbia, S.C.); Arcos, (Pittsburgh, Pa.) and TransWorld Chemicals (Rockville, Md.).

Material and Methods, Reagents, and Antibodies.

The anti-CD11b monoclonal antibody (mAb) 44a (an immunoglobulin G (IgG)2a (IgG2a) isotype) [73], the heterodimer-specific mAb 1B4 (IgG2a) [74,75], the activating anti-CD18 mAb KIM127 (IgG1) [61] and the anti-CD11bmAb ED8 (IgG1) [76] were from ATCC. The activating anti-CD18 mAb 24(IgG1) [59] was obtained from Abcam, the activating anti-CD11b mAb ED7(IgG1) [76] was from Sigma-Aldrich, the activating anti-CD18 mAb M18/2(IgG2a) [67] was from ebiosciences, the blocking anti-CD11b mAb OX42(IgG2a) [77] was obtained from Millipore and the isotype controlantibodies clone X40 (IgG1) and clone X39 (IgG2a), fluoresceinisothiocyanate (FITC)-conjugated mAb A85-1 (rat anti-mouse IgG1),FITC-conjugated R19-15 (rat anti-mouse IgG2a), FITC-conjugated goatantibody against mouse immunoglobulin, rat antibody against mouse GR-1(GR1-FITC), and phycoerythrin (PE)-conjugated rat antibody against mouseCD11b were obtained from BD Pharmingen. M1/70, a rat mAb against mouseCD11b (IgG2b) [78] was from the monoclonal antibody core at Universityof California, San Francisco (UCSF). Human fibrinogen (depleted ofplasminogen, von Willebrand factor, and fibronectin) was from EnzymeResearch Laboratories, bovine serum albumin (BSA) was from Sigma, LPS(O111:B4) was from Invivogen, and phorbol-12-myristate-13-acetate (PMA)was from Cell Signaling. Maxisorp and Highbind 384-well plates wereobtained from Nalgene and Corning, respectively. Non-fat milk wasobtained from BioRad. All cell culture reagents were from InvitrogenCorp. and Mediatech. Fetal bovine serum (FBS) was purchased from AtlantaBiologicals, Inc. The antibiotic G418 was purchased from Invivogen.

The wild type Sprague-Dawley (SD) rats were purchased from HarlanLaboratories. Animal care and procedures were approved by theInstitutional Animal Care and Use Committee (IACUC) and were performedin accordance with institutional guidelines.

Cells and Cell Lines.

K562 cells stably transfected with plasmid encoding wild-type integrinCD11b/CD18 (K562 WT cells) have been described previously [49, 79] andwere maintained in Iscove's Modified Dulbecco's Medium (IMDM)supplemented with 10% FBS and G418 (0.5 mg/ml). The murine macrophagecell line (RAW 264.7 cells) was obtained from ATCC and the cells weremaintained in DMEM supplemented with 10% heat-inactivated FBS accordingto the manufacturer's instructions. The primary rat peritonealmacrophages were isolated from the wild type Fisher 344 rats that hadbeen previously injected with 5 mL of Brewer's thioglycolate broth(Sigma-Aldrich). Macrophage purity was directly analyzed by singlechannel flow cytometry using rat macrophage specific monoclonalantibodies ED1 (AbD Serotec, Raleigh, N.C.), WT.5 (BD Biosciences) andHis36 (BD Biosciences). Purity was typically >85%. Macrophages were usedin assays immediately upon isolation.

Cell Adhesion Assays.

Cell adhesion assays using K562 WT, murine RAW 264.7 cells and theprimary rat macrophages were performed as previously described and usedimmobilized fibrinogen as the ligand of CD11b/CD18 [49]. A stocksolution of the leukadherin family of small-molecule agonists LA1, LA2,and L15 was prepared by dissolving each compound in DMSO at aconcentration of 10 mM. The final concentration of DMSO in the assay wasapproximately 1%. Cells were suspended in serum-free DMEM and incubatedin the presence of various agents in ligand-coated wells in a 384-wellplate for 10 minutes at 37° C. LA1, LA2, and LA15 were used at a finalconcentration of 15 pM each and the mAbs were used at a finalconcentration of 20 □g/mL each in the assays (LA2 and LA15 are shown inFIG. 10). The assay plates were then gently inverted and kept in theinverted position for 30 minutes at room temperature to dislodge thenonadherent cells. The remaining adherent cells were fixed using 4%formaldehyde and quantified by imaging microscopy as previouslydescribed (22, 63). Assays were performed in triplicate wells. Datareported are from one of at least three independent experiments.

Wound Healing Assay.

Murine RAW 264.7 macrophage cells were grown in DMEM 10% FBS medium inT75 flasks to 70% confluence. The cells were detached by trypsintreatment for 10 minutes at 37° C. and evenly reseeded in 24 well platesand allowed to attain >70% confluency. A scratch was introducedhorizontally across the well using 200 □L tip applying even forcethroughout the etching and holding the tip at approximately an 80 degreeangle. The wells were subsequently washed and fresh medium containingvarious treatment groups was introduced. The wound was imaged using 10×objective. The cells were allowed to grow for 48 hours and the wound wasreimaged at the same field after 48 hours. The average extent of woundclosure was evaluated by measuring the width of the wound. The areahealed was analyzed using ImageJ software and is expressed as percentagehealing after 48 hours.

Immunofluorescence Microscopy.

To examine localization and clustering of CD11b/CD18 on the cellsurface, 1×104 K562 CD11b/CD18 cells were suspended in serum-free IMDMand incubated in the presence of control DMSO (1%), leukadherins LA1,LA2, or LA15 (15 μM each), mAb KIM127 (1:100 dilution of ascites), mAb24 (20 □g/mL) or Mn2+ (1 mM) for 1 hour at 37° C., as describedpreviously [55, 72]. To visualize changes upon ligand binding,fibrinogen (50 μg/mL) was also added to the cell suspension media in asecond set of incubations. The cells were fixed in suspension andincubated with mAb 184, which is specific for CD11b/CD18, followed byAlexa Fluor 488-conjugated goat antibody against mouse Ig (Sigma).Fluorescence images were recorded with a Leica DMI16000 deconvolutionmicroscope using an HCX APO 40×/0.75 DRY objective with a DCF360FXcamera and with Leica LAS-AF software. A 3-dimensional representation ofCD11b/CD18 fluorescence intensity and the analysis of the number ofCD11b/CD18 clusters per cell was performed using ImageJ software asdescribed [80]. Clustering determination was carried out by countingindividual fluorescent peaks that were projected from the basal to theapical side of the cell and were at least 50% above the base linelevels. The images presented are representative of at least 20 cellsanalyzed for each condition from at least three independent experiments.

Western Blotting Analysis.

K562 WT cells were incubated with LA1, LA2, LA15 (15 μM), or fibrinogen(200 μg) in serum-free medium for 1 hour at 37° C. Cell lysates wereresolved on 4-12% NuPAGE Bis-Tris SDS-PAGE gels using MES running bufferand transferred to a polyvinylidene difluoride (PVDF) membrane(ThermoScientific) using established protocols. Membranes were incubatedwith a 1:1,000 dilution of the anti-phospho-protein antibodies (antiphospho-p44/42 MAPK (pERK1/2) antibody (Thr202/Tyr204, Cell Signaling)or anti-phospho-SAPK/JNK (pJNK) antibody (Thr183/Tyr185, Cell Signaling#81E11)), or with an antibody against either total ERK1/2 (p44/42 MARK(Erk1/2), Cell Signaling #137F5) or total JNK (SAPK/JNK, Cell Signaling#56G8) and developed according to the manufacturer's instructions(ThermoScientific). Proteins were quantified by densitometry usingImageJ software. Data presented are representative of two to threeindependent experiments.

Flow Cytometry.

Flow cytometric analyses of K562 WT cells were performed according topublished protocols [55, 57, 81]. Briefly, cells were suspended in theassay buffer (Tris-buffered saline (TBS) containing 1 mM of ions Ca2+and/or Mg2+ and 0.1% BSA). Cells (5×105) were incubated with primary mAb(1:100 dilution of IB4 ascites or the isotype controls, 1:50 dilution ofKIM127 ascites or 15 □g/mL of mAb 24) in 100 DL buffer in the absence orpresence of 20 □M agonist LA1 on ice (except for mAbs KIM127 and 24 andthe relevant isotype controls, where incubations were performed at 37□C) for 30 minutes. Subsequently, the cells were washed three times withthe assay buffer and incubated with goat anti-mouse-APC (1 □g/ml,Invitrogen) for 20 minutes at 4° C. Cells were washed twice with theassay buffer and analyzed using FACSCaliber flow cytometer (BDBiosciences, CA), counting at least 10,000 events. Data was analyzedusing the CellQuest software (BD Biosciences). Data shown is from one ofat least three independent experiments.

Balloon-Induced Arterial Injury in Rats.

All surgeries were performed under anesthesia by isoflurane (Baxter).Activating mAb ED7 (4 mg/kg/d), the isotype control mIgG1 (4 mg/kg/d,Rockland) and the leukadherin LA1 (1 mg/kg/d) were each administeredintra peritoneally (i.p.) in saline daily until the end of theexperiment. Balloon injury in the right iliac artery was inflicted witha 2F Fogarty catheter (Baxter) adapted to a custom angiographic kit(Boston Scientific, Scimed) [82]. An aortotomy in the abdominal aortawas made to insert a catheter to the level of the right iliac artery.The balloon was inflated to 1.5 to 1.6 atmospheres and retracted to thearteriotomy site three times. The aortic excision was repaired witheight sutures, The abdominal cavity was closed by planes with aninterrupted suture pattern. Arterial specimens were collected 21 daysafter injury and fixed in 4% formalin-PBS (Sigma-Aldrich) for 5 minutesand analyzed by histology and immunostaining. Neointima were measured inH&E stained slides using ImagePro.

Flow cytometric analyses for quantitation of arterial macrophages ininjured arteries.

Agonist treated and control balloon-injured Fisher 344 rats weresacrificed 7 days post surgery at the onset of inflammation. The injuredand non-injured iliac arteries were microdissected and digested withCollagenase/Elastase mix (Worthington Biochemical, NY) in DMEMcontaining 2% FBS for 2 hours at 37° C. The resulting single cellsuspension was washed with cold PBS containing 2% FBS and filteredthrough 70 □M sieve. Cells were re-suspended cold PBS containing 2% FBSat a concentration of 106 cells per 100 μl and stained with biotinylatedanti-rat CD11b antibody (clone WT.5, BD Biosciences) for 1 hour at 4° C.Subsequently, cells were washed and incubated with APC-Streptavidin for30 minutes at 4° C. Stained cells were fixed with formalin 4% in PBS for10 minutes. Cells were washed twice with the assay buffer and analyzedusing FACS II Canto cytometer and the data were analyzed using FlowJo(Tree Star Inc.) software. Data presented is from 4-6 independentsamples/group.

Statistical Analysis.

Data were analyzed with GraphPad Prism and compared with the Student's ttest or by one-way analysis of variance (ANOVA) with posthoc analysis,where appropriate. P<0.05 was considered statistically significant.

Flow Chamber Assay.

The flow chamber assay was performed as described in literature (Chen,J. F., Salas, A. & Springer, T. A. Bistable regulation of integrinadhesiveness by a bipolar metal ion cluster. Nat. Struct. Biol. 10,995-1001 (2003)). A polystyrene Petri dish was coated with a 5 mmdiameter, 20 μL spot of 20 μg/mL purified h-ICAM-1/Fc (R&D) or 20 μg/mLFibrinogen in coating buffer (PBS, 10 mM NaHCO₃, pH 9.0) for 1 hour at37° C., followed by 2% BSA in coating buffer for 1 hour at 37° C. toblock nonspecific binding sites. Transient tansefected 293T cells werewashed twice with wash buffer (20 mM Hepes, 150 mM NaCl, pH 7.4, 5 mMEDTA/0.5% BSA), subsequently once with HBS containing 1 mM Ca²⁺ and 1 mMMgt* (HBS⁺⁺), and finally resuspended at the concentration of 5×10⁶ /mLin HBS⁺⁺ (Ca²⁺ and Mg²⁺-free HBS, 0.5% BSA) and kept on ice. Cells wereincubated in 2% DMSO with or without 25 μM LA1 at 37° C. for 30 minutes.And subsequently infused in the flow chamber using a Harvard apparatusprogrammable syringe pump. Cells were allowed to settle down for 5minutes, and accumulate for 30 seconds at 0.3 dyn/cm² and 10 seconds at0.4 dyn/cm². Then, shear stress was increased every 10 seconds from 1dyn/cm² up to 32 dyn/cm², in 2-fold increments. The number of cellsremaining abound at the end of each 10-sec interval were determined.Rolling velocity at each shear stress was calculated from the averagedistance traveled by rolling cells in 3 seconds. Rolling adherent cellswere defined with a velocity more than 1 μm/s. Adhesive behavior ofvehicle-, Mn2+ or LA-treated CD11b/CD18 transfectants under the wallshear stress is shown.

Results

Integrin agonists have several advantages over antagonists. Researchwith antagonists over last several years has shown them to besuboptimal. First, it has been showed that suppressing leukocyterecruitment with antagonists requires occupancy of >90% of activeintegrin receptors [2], usually requiring high levels of blockingantibodies in vivo. Second, complete blockade of cell surface-expressedCD11b/CD18 even with antibodies is difficult due to availability of alarge mobilizable intracellular pool of CD11b/CD18 [30, 31]. Third,several other antagonists, such as ligand-mimetic neutrophil inhibitoryfactor (NIF) [67] and recombinant αA-domain [68], were effective inanimal models but their large size and immunogenicity preclude their useas a therapeutic agent. Recombinant NIF (UK-279276) failed in clinicaltrials. Likewise, peptides derived from either anti-CD11b/CD18antibodies or CD11b/CD18 ligands are not very efficacious in blockingligand binding in vitro [69], perhaps owing to their improperconformation in solution or to their small size relative to theligand-binding region on CD11b/CD18. Finally, many antagonisticantibodies (such as rhuMAb CD18, anti-CD18 LeukArrest (Hu23F2G) andanti-ICAM1 mAb Enlimomab (R6.5)) failed in treatinginflammatory/autoimmune diseases in several clinical trials [28, 29] andβ2 integrin blockers have also shown unexpected side effects and havehad to be withdrawn from the market [33].

Chemical and biological agonists of integrin CD11b/CD18 enhance celladhesion. It was proposed that integrin activation could be a novelmechanism for the development of next-generation anti-inflammatorytherapeutics that reduce inflammatory cell migration and recruitment byenhancing, rather than reducing, cell adhesion [49, 55]. To that end,Applicant recently described novel small molecule agonists ofCD11b/CD18, which Applicant termed leukadherins [55], that Applicantidentified using a cell-based high-throughput screening assay [49, 50].Leukadherin-1 (LA1) showed high-affinity for CD11b/CD18 and increasedcell adhesion by binding to an allosteric pocket of the ligand-bindingαA-domain (also known as al-domain) of CD11b and stabilizing it in anactive form [55—see FIG. 1B]. Applicant also identified additionalanalogs of LA1 that showed similar activity (including LA2 and LA15,FIG. 10). Similarly, a number of biological agonists of CD11b/CD18 (andother b2 integrins), in the form of activating antibodies, have beendeveloped over the years by others, including anti-CD18 monoclonalantibody (mAb) KIM127 [83] and anti-CD18 mAb 24 [59].

To determine how the two types of agonists—chemical (LA1) and biological(mAbs KIM127 and 24)—affect ligand binding by CD11b/CD18, a head-to-headcomparison was performed of LA1, mAb KIM127, and mAb 24 for theirrelative abilities to increase adhesion of CD11b/CD18 to immobilizedphysiologic ligand fibrinogen using K562 cells stably expressing wildtype CD11b/CD18 (K562 WT). K562 WT cells constitutively express wildtype CD11b/CD18 in a low affinity state (as in normal leukocytes) [66,79], thus, providing an excellent system to examine the level ofintegrin activation afforded by various agents [55, 66]. K562 WT showedminimal adhesion to immobilized fibrinogen in the presence ofphysiologic divalent cations (Ca2+ plus Mg2+, each at 1 mM). The knownnon-selective integrin activator Mn2+ [84, 85] significantly enhancedcell adhesion, increasing it to a maximal level, which was significantlyblocked to basal levels by a blocking anti-CD11b antibody 44a that bindsto the αA-domain [73]. Similarly, agonists LA1 and activating mAbsKIM127 and 24 significantly increased K562 WT cell adhesion toimmobilized fibrinogen, as compared to the basal Ca2+ plus Mg2+condition. However, it was found that agonist mAb 24 produced a lowerincrease in the level of cell adhesion as compared to the other twoagonists—LA1 and KIM127. In all cases, the adhesion of K562 WT toimmobilized fibrinogen was also significantly reduced by the anti-CD11bblocking mAb 44a, further confirming that the increased cell adhesion byall of these agonists was mediated by CD11b/CD18. As Applicant has shownbefore, this agonistic effect of leukadherins is not limited to a singlecompound, as additional leukadherins LA2 and LA15 similarly enhancedCD11b/CD18-mediated cell adhesion (FIG. 11). FIG. 11 is a histogramshowing percentage adhesion of K562 WT cells to immobilized fibrinogenin the presence of physiologic 1 mM Ca²⁺ and 1 mM Mg²⁺ ions (Control),the non-selective integrin agonist Mn²⁺ (1 mM), or the agonists LA2 (15μM) and LA15 (15 μM) in the absence or presence of blocking anti-CD11bantibody 44a (1:100 dilution of ascites fluid). Data shown are mean±thestandard error of the mean (SEM) (n=3 to 6 replicates per condition) andare from one of at least three independent experiments. ***p<0.0001.

Next, to examine if the chemical and biological agonists of CD11b/CD18have a similar effect on ligand binding by CD11b/CD18 from differentspecies, the murine macrophage cell line RAW 264.7 and the primary ratmacrophages that have constitutive CD11b/CD18 surface expression wereused. The small molecule compound LA1 binds to an allosteric pocket inthe αA-domain of CD11b (referred to as Socket for Isoleucine (SILEN)[86] in CD11b or IDAS in CD11a [87]) that is highly conserved acrossvarious species [88], suggesting that LA1 should be able to activateCD11b/CD18 from various species. However, the biological agonists (e.g.;activating mAbs) are highly species-selective. Therefore, awell-characterized anti-mouse activating antibody (M1812) [67] was usedwith the murine RAW 264.7 cells and the anti-rat anti-CD11b activatingantibody ED7 [70, 76] was used with the primary rat macrophages. Theanti-mouse CD18 mAb M18/2 is a non-blocking antibody that has beenreported to simulate CD11b/CD18-dependent cell adhesion and rosetting[67-69]. The anti-rat CD11b antibodies ED7 (and another mAb ED8) havebeen shown to enhance CD11b/CD18-dependent granulocyte adhesion andhomotypic aggregation under certain conditions, suggesting that theyactivate CD11b/CD18 [70].

The RAW 264.7 cells showed minimal level of adhesion to immobilizedfibrinogen in the presence of physiologic 1 mM Ca2+ and Mg2+, whichsignificantly increased (to maximal levels) with the agonist Mn2+. Aswith K562 WT cells, agonists LA1 and the activating mAb (M18/2)significantly and similarly increased the level of cell adhesion, ascompared to the basal condition. Furthermore, in both cases, addition ofblocking anti-CD11b mAb M1/70 [78] eliminated the increase incell-adhesion due to the two agonists, again confirming that CD11b/CD18mediated the increased cell adhesion by both types of agonists.Similarly, the primary rat macrophages showed almost no adhesion toimmobilized fibrinogen in the presence of non-activating, physiologicCa2+ and Mg2+ ions, but bound significantly more upon activation withMn2+. Again, both types of agonists—the chemical LA1 and the activatingmAb ED7—significantly enhanced the level of macrophage adhesion as well,as compared to the non-activating condition, and, in both cases,addition of blocking anti-CD11b mAb OX42 [77] greatly reduced theeffects of the two agonists, confirming that the increased ratmacrophage cell adhesion by both agonists was mediated by CD11b/CD18.Surprisingly, ED8, which has been reported as an agonist similar to ED7and having an overlapping epitope with ED7 [70, 76], did not produce anysignificant enhancement in the adhesion of the primary rat macrophagesto immobilized fibrinogen (data not shown), showing that ED7 is astronger agonist than ED8. Taken together, these results show thatselective agonists of the integrin CD11b/CD18, be it chemical orbiological, enhance CD11b/CD18-dependent cell adhesion. Additionally, itshows that while biologics have strong species dependence, leukadherinsare equally effective on human, murine and rat CD11b/CD18.

Activation of CD11b/CD18 Reduces Macrophage Cell Migration andWound-Healing.

Wound-healing assays are routinely used as a classic method for studyingcell migration and to assess the effects of various perturbations of theunderlying biological processes on Such a key cellular function [89,90]. Macrophages play a significant role in wound-healing [91] andconfluent monolayers of macrophages, when artificially wounded orscratched with a pipette tip, respond to the disruption of cell-cellcontacts by healing the wound through a combination of proliferation andmigration [89]. Macrophages and other leukocytes use CD11b/CD18dependent cell adhesion (as well as other b2 integrins) to migrate overtwo-dimensional surfaces [92]. Applicant has previously shown thatCD11b/CD18 agonist LA1, by freezing integrin in a ligand-bound state,impairs neutrophil chemotaxis [55]. However, how LA1 compares to abiological agonist of the integrin CD11b/CD18 in a head-to-headcomparison is not known. Here, a murine macrophage cell-basedwound-healing assay was used to compare the relative efficacy of LA1with a biological agonist of CD11b/CD18 (the anti-CD18 activating mAbM18/2) using the RAW 264.7 cells [93]. The RAW 264.7 cells were platedto confluence and the cells were ‘wounded’ by scraping a marked locationon the plate with a pipette tip.

As shown in FIGS. 4A and 4B, stimulation with either lipopolysaccharide(LPS) or with the phorbol ester phorbol-12-myristate-13-acetate (PMA)significantly induced the migration of macrophages back into the woundarea, as compared to the unstimulated cells (DMSO). The plot in FIG. 4Ashows percentage of wound area that is healed via migration of RAW 264.7macrophages into the wound under various conditions 48 hours after thepipette tip induced injury. **p<0.001, ***p<0.0001, ns=not significant.For FIG. 4B, RAW 264.7 cells were plated in 24-well tissue cultureplates (2×10⁶ cells/well) and allowed to adhere in complete media for 12hours. The cell monolayers were treated with vehicle (DMSO, agonist LA1(15 □M), activating antibody M18/2 (20 □g/mL) and with LPS (100 ng/mL)or PMA (100 nM) that accelerate cell migration and wound-healing, for 1hour prior to wounding with a pipette tip. Furthermore, cell monolayersin additional wells were treated with LPS (100 ng/mL) or PMA (100 nM) inthe presence of LA1 (15 □M) or M18/2 (20 □g/mL) to investigate whetherincreasing CD11b/CD18-dependent cell adhesion will impact thewound-healing process under these conditions. Subsequently, the woundedmonolayer cell cultures were incubated for 48 hours. Images of the cellsin culture were obtained immediately prior to (0 hours) and after thecompletion of the experiment (48 hours) using an inverted phase contrastmicroscope attached to a video camera.

Expectedly, treatment with the agonists LA1 and M18/2 alone showed nosignificant increase in the migration of macrophages into the woundarea, as compared to unstimulated cells (DMSO). However, both CD11b/CD18agonists significantly, and to a similar extent, reduced the migrationof LPS- or PMA-stimulated macrophages. Additionally, the compounds LA2and LA15 similarly reduced the migration of LPS-stimulated macrophages(FIG. 12), showing that the leukadherins family of compounds has asimilar effect on macrophage migration. FIG. 12 is a histogram showingpercentage of wound area that is healed via migration of RAW 264.7macrophages into the wound under various conditions 48 hours after thepipette tip induced injury. RAW 264.7 cells were plated in 24-welltissue culture plates (2×10⁶ cells/well) and allowed to adhere incomplete media for 12 hours. The cell monolayers were treated withvehicle (DMSO), agonist LA2 (15 □M), or agonist LA15 (15 □M) and withLPS (100 ng/mL) for 1 hour prior to wounding with a pipette tip.Furthermore, cell monolayers in additional wells were treated with LPS(100 ng/mL) in the presence of LA2 (15 □M) or LA15 (15 □M) toinvestigate whether increasing CD11b/CD18-dependent cell adhesion willimpact the wound-healing process under these conditions. Subsequently,the wounded monolayer cell cultures were incubated for 48 hours. Imagesof the cells in culture were obtained immediately prior to (0 hours) andafter the completion of the experiment (48 hours) using an invertedphase contrast microscope attached to a video camera and quantitatedwith ImageJ. Representative data shown here are from a triplicate wellexperiment from one of at least two to three independent experiments.**p<0.001, ***p<0.0001. These results show that integrin CD11b/CD18activation via either type of agonist equally impairs the migratorycapacity of macrophages.

Activating Antibodies, but not LA1, Induce Integrin Clustering andIntracellular Signaling.

Next, it was evaluated if there were any significant differences in theintegrin binding mediated signaling that is induced by the two types ofagonists. While the activating antibodies and leukadherins both enhanceCD11b/CD18-dependent cell adhesion and reduce cell migration, it ispossible that integrin activation can induce undesirable intracellularsignaling, such as those that have been described for the integrinantagonists, which can limit their effectiveness and utility in theclinic [94-100]. For example, aIIbb3 and aVb3 antagonists induceoutside-in signaling [94, 96]. Similarly, CD11b/CD18 ligation withligands or blocking antibodies induces CD11b/CD18 clustering [28] andoutside-in signaling via activation and phosphorylation of mitogenactivated protein kinases (MAPKs), including the c-Jun NH2-terminalkinase (JNK), the p38 MAPK and the extracellular signal-regulated kinase(ERK) [28, 33, 71, 101], thereby up-regulating the pro-inflammatoryNF-kB signaling [29, 32, 102]. Additionally, both inactive and activeCD11b/CD18 can be induced to form macro clusters, but the two types ofmacro clusters transduce different intracellular signals [28],suggesting that an analysis of both clustering and intracellularsignaling is needed to fully study the effects of the two types ofCD11b/CD18 agonists.

Therefore, to investigate, a combination of confocal microscopy (tostudy clustering) and western blotting assays (to study intracellularsignaling) using K562 WT cells were used. First, CD11b/CD18 cell-surfacedistribution and macro clustering was studied under various conditionsusing confocal microscopy. K562 WT cells in suspension were incubatedwith various agonists, fixed with paraformaldehyde and the surfaceexpressed CD11b/CD18 was fluorescently stained with anti-CD11b/CD18 mAb1B4. The results are presented in FIGS. 5A and 5B. For FIG. 5A, cellsuspensions were incubated for 45 minutes at 37° C. with DMSO (vehicle),non-selective agonist Mn²⁺+(1 mM), agonist LA1 (15 uM), activating mAbKIM127 (1:100 dilution of ascites fluid), or the activating mAb 24 (20μg/mL) in the absence (top panel, −Fg) or the presence of the ligandfibrinogen (50 μg/mL) (bottom panel, +Fg) and the cells were fixed withparaformaldehyde (4%) prior to labeling the surface expressed CD11b withanti-CD11b/CD18 mAb IB4 and imaged. The fluorescence images shown arerepresentative of three independent experiments. White scale barrepresents 25 μm. Also shown below each image is a 3D representation ofCD11b fluorescence intensity for selected cells, analyzed in ImageJ InFIG. 5B, data shown are mean±SEM from >40 cells per condition andfrom >3 independent experiments for each condition. ***p<0.0001, ns=notsignificant.

K562 WT cells showed uniform distribution of CD11b/CD18 on the cellsurface, with minimal macro clustering, in the absence of any stimulus,ligands, agonists or antibodies (FIG. 5A, top panel, DMSO). As has beenreported before, incubation of cells with ligand fibrinogen (Fg)resulted in significant CD11b/CD18 macro clustering (FIG. 5A, bottompanel, DMSO) when quantitated using ImageJ [80] (FIG. 5B). The agonistMn2+, which has been shown to also induce CD11b/CD18 clustering [28],reproduced the clustering phenotype here (FIGS. 5A and 5B). Incubationof cells with the activating antibodies KIM127 and mAb 24 also resultedin significant CD11b/CD18 macro clustering on the cells surface and thisincreased level of integrin macro-clustering did not significantlychange upon addition of the physiologic ligand fibrinogen, suggestingthat activating anti-CD11b/CD18 antibodies induce CD11b/CD18 activationand clustering even in the absence of a ligand. Previous studies havealso shown that other activating anti-CD11b antibodies, including VIM12(which binds near the extracellular C-terminal region of CD11b [56]) andthe full-length immunoglobulin (IgG) as well as the Fab-fragment ofLFA-1/2 (which binds the EGF3 domain of CD18 [57, 58]), induceCD11b/CD18 clustering [28, 71]. However, incubation of the cells withthe agonist LA1 alone did not induce any significant CD11b/CD18 macroclustering (FIG. 5A, top panel, LA1) in the absence of ligandfibrinogen, but did so in the presence of fibrinogen [55]. Theleukadherins LA2 and LA15 showed similar results (FIGS. 13A and 13B). InFIG. 13A, cell suspensions were incubated for 45 minutes at 37° C. withDMSO (vehicle), non-selective agonist Mn²⁺ (1 mM), the agonist LA2 (15uM) or the agonist LA15 (15 uM) in the absence (top panel, −Fg) or thepresence of the ligand fibrinogen (50 μg/mL) (bottom panel, +Fg) and thecells were fixed with paraformaldehyde (4%) prior to labeling thesurface expressed CD11b with anti-CD11b/CD18 mAb IB4 and imaged. Thefluorescence images shown are representative of three independentexperiments. White scale bar represents 25 μm. Also shown below eachimage is a 3D representation of CD11b fluorescence intensity forselected cells, analyzed in ImageJ. For FIG. 13B, data shown aremean±SEM from >40 cells per condition and from >3 independentexperiments for each condition. ***p<0.0001, ns=not significant. Thesedata in FIGS. 5A-5B and 13A-13B show that the two types of integrinagonists have very different effects on the integrins on thedistribution and clustering of CD11b/CD18 on the surface of live cells.

Second, an effect of integrin ligation, macro clustering andredistribution in the plasma membrane is the triggering of outside-inintracellular signaling in cells via various MAPKs [103]. Triggering ofsuch MAPK signals also rapidly stimulates transcription and secretion ofa variety of pro-inflammatory cytokines and chemokines in leukocytes[29]. To test, two such MAPK pathways were investigated—the ERK pathwayand the c-Jun NH2-terminal kinase (JNK) pathway—by measuring therelative levels of phosphorylated ERK (pERK) and phosphorylated JNK(pJNK) using western blotting. K562 WT cells incubated with vehicle(DMSO) alone showed minimal ERK and JNK activation (FIGS. 6A-6D) ascompared to cells that were incubated with ligand fibrinogen or wereactivated with the protein kinase C (PKC) agonist phorbol ester PMA(positive control, [104]). In FIGS. 6A-6B, immunoblot results from twoindependent experiments were quantified using ImageJ software and ratioof phosphorylated protein to total protein was determined. Histogramsshowing this quantitation for each set are also shown. Data shown aremean±SEM. *p<0.05, ***p<0.0001, ns=not significant. In FIGS. 6C-6D,immunoblot results from two independent experiments were quantified andare also shown, as in 6A and 6B.

Incubation of cells with agonist Mn2+ or the activating antibodiesKIM127 or mAb 24 showed a significant activation of both ERK and JNK.Previously published studies have also shown that incubation ofCD11b/CD18 expressing cells with either the agonist Mn2+ [101], blockingmAbs, ligand ICAM- or other anti-integrin activating mAbs [71],similarly induces ERK phosphorylation and MAPK signaling. Incubation ofthe cells with the agonist LA1 alone did not induce any significant pERKor pJNK in the absence of ligand fibrinogen. Additionally, theleukadherins LA2 and LA15 showed similar results (not shown). CD11b/CD18ligation with fibrinogen showed high levels of both pERK and pJNK underall conditions, suggesting that such MAPK signaling may mimic aligand-bound state of the cell. Thus, it is concluded that CD11b/CD18binding to activating antibodies mimics its ligand-bound state andinduces intracellular MAPK signaling, whereas CD11b/CD18 binding toleukadherins does not. It is worth noting that leukocytes from knock-inanimals expressing constitutively active integrins CD11a/CD18 (LFA-1) or0407 do not show MAPK signaling in the absence of ligand [105-107],similar to the results obtained with the leukadherins.

Collectively, these results show that while the two types of integrinagonists may have some common functional effects on CD11b/CD18expressing cells (that they enhance ligand binding and cell adhesion),they can also have significant differences that show that one can bemore beneficial for in vivo use over the other.

LA1 does not induce global conformational changes in CD11b/CD18. Ligandbinding induces extension of the integrin heterodimer and large, globalconformational changes leading to outside-in signaling [28, 69, 71].Binding of activating antibodies also induces global conformationalchanges in the integrin heterodimer and such large conformationalchanges mimic those induced upon ligand binding [71, 72]. For example,activating antibody KIM127 recognizes a region in the EGF2 domain ofCD18 that is buried in the inactive, bent conformation of the integrinCD11/CD18 heterodimers and stabilizes an extended conformation uponbinding [57, 61, 66], which also leads to the separation of thecytoplasmic tails of the heterodimer [71, 108]. Thus, mAb KIM127 isoften used as a reporter for the extended conformation of P2 integrins[70, 79, 83]. Similarly, mAb 24 binds to an activation-dependent epitopein the αA domain that mimics the ligand-bound conformation of thevarious CD11/CD18 heterodimers [59]. To determine the mechanistic basisfor the lack of outside-in signaling by LA1 in the treated cells, theextent of the conformational changes caused by LA1 binding tocell-surface expressed CD11b/CD18 in K562 cells was investigated, usingmAbs KIM127 and 24 as reporters of large/global conformational changes.Incubation of K562 WT cells in physiologic buffer alone showed basallevel of KIM127 and mAb 24 binding, as measured by flow cytometry (FIGS.7A and 7B). The binding by isotype IgG1 control antibody (15 ug/mL) isshown in the topmost panels. Cells were incubated with mAbs KIM127 or 24in the absence of agonists (Control), in the presence of LA1 (15 uM) orMn²⁺ ions (1 mM). Data shown are representative of at least threeindependent experiments.

Expectedly, addition of integrin agonist Mn2+ ions was sufficient toinduce maximal increase in the amount of bound KIM127 and mAb 24,mimicking the ligand binding induced global changes in the CD11b/CD18heterodimer as has been reported in the literature [59, 70, 79, 83].However, incubation of these cells with small molecule agonist LA1produced a very small increase in the KIM127 binding (FIG. 7B),indicating that the LA1-mediated DA activation does not expose theKIM127 epitope in the EGF2 of CD18 and showing that LA1 binding leads todomain-limited conformational changes, but not large globalconformational changes in the CD11b/CD18 heterodimer on live cellsurfaces. The global conformational changes in integrins triggercytoplasmic leg-separation and generate outside-in signaling [71, 108].Similarly, LA1 binding showed a small increase in the binding of thesecond conformational reporter probe, the mAb 24, over the basal levelof binding by mAb 24 to cell surface-expressed CD11b/CD18. In bothcases, the level of reporter probe binding to CD11b/CD18 in the presenceof LA1 was substantially less as compared to their binding in thepresence of ligand-mimetic agonist Mn2+ ions.

While LA1 significantly reduces mechanical vascular injury in rats, theanti-CD11b activating antibody ED7 is ineffective. Percutaneous coronaryintervention (PCI) is one of the most effective methods to unblockoccluded arteries and facilitates coronary revascularization in humans[109]. Despite significant technological advances in PCI, restenosis(re-narrowing) secondary to neointimal hyperplasia remains a majorcomplication limiting the success of coronary interventions in 5-25% ofpatients [110-112]. The recruitment of inflammatory macrophages andother leukocytes precedes neointimal hyperplasia [5]. The denudation ofendothelial cell lining at the site of mechanical vascular injury duringa PCI procedure leads to the deposition of fibrin and platelets, whereselective binding between the platelet cell surface receptor GP lb□ andleukocytic CD11b/CD18 leads to the migration and recruitment of theseinflammatory cells [113]. Indeed, in experimental models, it has beenshown that the genetic ablation of CD11b (CD11b−/− animals) or itsblockade (using biological antagonists) can reduce neointima formationafter angioplasty or stent implantation [5, 39]. Applicant has alsopreviously demonstrated that LA1 significantly reduces macrophageaccumulation at the site of mechanical vascular injury, resulting insignificantly reduced neointimal hyperplasia [55]. Here, thisexperimental arterial balloon injury model was used to examine therelative efficacy of the two types of agonists, in a head-to-headcomparison, on inflammatory responses in vivo. First, thedose-dependence of LA1 in this model system was determined. LA1 wasadministered at two different doses (0.05 or 1 mg/kg/d, intraperitoneal(i.p.)), or the vehicle (DMSO), in a saline solution to SD male rats 30minutes prior to injury and continued daily injections for the nextthree weeks and subsequently measured the extent of vascular injury viatissue histology (FIGS. 14A-14D). Data shown are mean+SEM. *p<0.05,**p<0.001. The injured arteries of LA1-treated rats developedsignificantly less neointimal hyperplasia (FIGS. 14B and 14C) ascompared to those of animals receiving vehicle alone (FIG. 14A).Additionally, LA1 showed a dose-dependent reduction in the extent ofballoon catheter-induced mechanical vascular injury, with the higherdose producing a more significant reduction in it as compared to thevehicle treated control.

Next, to determine whether either type of agonist (chemical LA1 andbiologic activating antibody) has a therapeutic advantage over theother, a head-to-head comparison was performed using this in vivo model.As the study involves use of rats as the experimental model system, theanti-rat CD11b activating mAb ED7 was chosen for comparison with LA1.Previous reports show that administration of ED7 reduced kidney andinflammatory bowel disease in rats [114, 115]. The mAb ED7 was obtainedwith high purity from ascites fluid according to published protocols[116]. Staining and flow cytometric analysis showed high degree ofbinding by the anti-CD11b mAb ED7, but not the control mAb mIgG1, to thepurified primary rat macrophages and a selective enhancement inmacrophage cell adhesion to immobilized fibrinogen by ED7 vs the controlmAb IgG1. To examine the effects of the two types of agonists onmigration and influx of inflammatory macrophages into injured arteriesin this model of vascular injury, LA1 or ED7 was administered to wildtype rats 30 minutes prior to balloon injury and continued daily for thenext 7 days. The dosage of ED7 used was based on the published studies[114, 115, 117]. Next, the arteries were analyzed to quantify the numberof infiltrated macrophages in the presence of agonists LA1 or ED7 andcompared it to vehicle treated animals. The arteries were harvested fromanimals 7 days after balloon injury and single cell suspensions wereanalyzed by flow cytometry (using anti-rat CD11b antibody WT.5) toquantitate CD11b+ macrophages in each sample. The flow cytometry data,shown in FIGS. 8A-8D, reflects the distribution of macrophages withinthe arterial wall and the surrounding adventitia. Data shown arerepresentative of at least 4-6 independent measurements. Data shown aremean±SEM. *p<0.05, **p<0.001, ns=not significant. Consistent withprevious findings, injured arteries of LA1 treated animals containedsignificantly less CD11b/CD18 macrophages than those in the controlvehicle treated rats. It was also found that ED7 treatment alsosimilarly and significantly decreased the amount of infiltratedmacrophages in the injured artery and in a dose dependent fashion (twodoses of 1 mg/kg/d and 3.3 mg/kg/d were tested), showing the doses ofED7 used here to be highly effective in vivo. These results show thatboth types of agonists can similarly reduce macrophage influx into thetissue.

Next, in order to examine the relative efficacy of LA1 and ED7, LA1 (1mg/kg/d) or ED7 (4 mg/kg/d) were administered to SD male rats 30 minutesprior to balloon injury and continued daily for the next 21 days. Anisotype control antibody (mIgG1, 4 mg/kg/d) was administered to thecontrol group of animals. Histochemical analysis of the injured ratarteries showed reduced neointimal thickening in ED7- and LA1-treatedanimals, as compared to the control mAb IgG1-treated group (FIGS.9A-9D). Data shown are mean±SEM. *p<0.05, ***p<0.0001, ns=notsignificant. However, only LA1 showed a significant reduction inneointimal hyperplasia (NIH), as compared to the control group and alsoshowed significantly less neointima formation as compared to the ED7group, showing that LA1 is more efficacious than the CD11b-activatingmAb ED7 in vivo in the rat balloon injury model. Together, these resultsshow that leukadherin LA1 dose-dependently reduces vascular injury (asmeasured by neointimal thickening) and shows higher efficacy thananti-CD11b activating mAb ED7 in vivo and thus, has a clear therapeuticadvantage.

Leukadherins are a family of small molecule compounds that are novelintegrin CD11b/CD18 agonists. Leukadherins increase CD11b/CD18-dependentcell adhesion, reduce leukocyte migration and inflammatory injury invivo. The in vitro and in vivo efficacy of leukadherins suggested thatmany agents that activate CD11b/CD18 and enhance cell adhesion could bedeveloped into pharmacologically useful therapeutics to treatinflammation. However, in this study, it was found that not all integrinagonists are equal and that, as compared to the small molecule chemicals(leukadherins), biological agonists of CD11b/CD18, such as anti-CD11bactivating antibodies, have additional effects on intracellularsignaling by CD11b/CD18 that can reduce their potential as therapeutics.Such effects have also been reported with a number of ligand-mimeticintegrin antagonists that have shown worsened clinical outcomes, perhapsdue to induction of outside-in integrin signaling [118, 119]. It isfound that CD11b/CD18 activation via both types of reagents (thechemical leukadherins and the biologic activating mAbs) increasesintegrin-mediated cell adhesion and decreases cell migration and woundhealing in vitro. However, unlike leukadherins, the biologicanti-CD11b/CD18 activating antibodies induced clustering of the cellsurface expressed CD11b/CD18 and the phosphorylation of keyintracellular signaling proteins, including the JNK and ERK MAP kinases,showing that CD11b/CD18 engagement with activating antibodies mimicsligand-bound state. FIGS. 6A-6D clearly show that while both types ofCD11b/CD18 agonists, the chemical LA1 and the activating antibodies,bind and allosterically activate the integrin receptors, the two typesof agonists clearly induce different outside-in signaling in cells inthe absence of a ligand (fibrinogen). FIGS. 6A-6D also show that at ahigh concentration of ligand, the two types of agonists do not interferewith the typical ligand-binding induced outside-in signaling, and suchsignaling is the same under both cases. However, clearly an activatingantibody alone is sufficient to induce significant outside-in signalingwhereas LA1 binding per se did not induce any such signaling. It isimportant to note here that fibrinogen becomes appreciable for leukocyteadhesion and activation only after surface deposition [120-122].Fibrinogen deposition on the endothelial surface exposes the crypticy390-396 residues in fibrinogen that is recognized by the integrinCD11b/CD18 on leukocytes for high-affinity engagement, which leads torecruitment of leukocytes from circulation into the injured arteries.Furthermore, conformation-specific antibody probes were used toillustrate a potential reasoning behind these observed differences insignaling between the two types of agonists. Leukadherin binding did notinduce large global conformational changes in the integrin CD11b/CD18,whereas the activating antibodies have been shown to induce such changesthat mimic a ligand-bound integrin [71, 72]. Thus, while bindingactivating antibodies induce ligand-mimetic outside-in signaling incells, binding of Ieukadherins seems to induce more modest, localchanges in the integrin, which are likely not enough to induceoutside-in signaling in cells (FIGS. 9A-9D). It has also been previouslyreported in literature that constitutively active integrin mutants,where the ligand-binding □A-domain of CD11b is locked in its activeconformation, do not induce intracellular signaling [72]. Similarly,knock-in mice expressing constitutively active integrin mutants (□4□7[106], CD11a/CD18 [107]) do not show any increase in outside-insignaling in leukocytes expressing constitutively active integrins. Thisshows that □A-domain activation, by itself, is likely not sufficient forinducing ligand-binding mimetic conformational changes and outside-insignaling, whereas binding of large, antibody agonists, certainlyinduces global conformational changes in integrins, thereby producingligand-binding mimetic outside-in signaling. Thus, it further shows thatthe use of biologic activating antibodies can have additional unforeseenconsequences, while the chemical leukadherins seem to have limitednegative effects on leukocyte function. Additionally, when compared in ahead-to-head study in a vascular injury model in vivo, while both typesof agents similarly reduced the influx of inflammatory macrophages intothe injured arteries, leukadherin LA1 dose-dependently reduced vascularinjury and showed significantly higher efficacy than the anti-CD11bactivating antibody ED7, showing that this small molecule agonist ofCD11b/CD18 has a clear therapeutic advantage over the biologicactivating antibody. However, more detailed mechanistic studies in thefuture will also be needed to provide more insights into the differencesbetween the in vivo functions of the two types of agonists. Takentogether, the data presented herein shows that leukadherins, which arenewly discovered novel small molecule agonists of CD11b/CD18, areeffective anti-inflammatory agents in vivo, and can be developed intonovel therapeutics, whereas biological agonists of CD11b/CD18, in theform of anti-CD11b/CD18 activating antibodies, are sub-optimal and canrequire significant amount of optimization before they are ready for invivo use.

Over the last more than 15 years, there have been at least threepublished reports on the use of anti-CD11b activating antibody ED7 inreducing inflammatory injury in vivo [114, 115, 117]. Thus, it was quitesurprising to find that it was not more effective in reducing theballoon catheter induced vascular injury in rats here. There could beseveral reasons for this lack of efficacy of ED7. First, it isconceivable that the dose of ED7 used here (4 mg/kg/d) was inadequate.It is not believed that this was an issue here because it has previouslybeen shown (in multiple studies) that similar doses of ED7 effectivelyreduced—a) the mobilization of leukocytes into the peritoneal cavityafter thioglycollate-injection [123], b) intestinal damage in a model ofacute colitis [115], and c) structural and functional injury in ratswith Adriamycin-induced nephrosis [114]. Previous literature also showsthat an even lower dose of ED7 (1-3 mg/kg) is effective even whenadministered every other day or even weekly [114, 115, 117] and thatsuch amounts of anti-CD11b antibodies are sufficient enough tocompletely coat circulating leukocytes in the blood within 1 hour afterinjection and maintain it for at least 48 hours [115, 124]. Activatingantibodies at such high level of integrin receptor binding on cells havealso been previously reported in literature to have an effect on ligandbinding—that they increase ligand binding at these doses [71].Therefore, it was chosen to use this “effective” reported ED7 dose inanimals with injured arteries to prevent inflammation and diseasesdevelopment. Second, ED7 is a mouse anti-rat antibody and one couldargue that immune-clearance by the rodent system limited its efficacy.However, as ED7 has previously been shown to have at least some in vivoefficacy in wild type rats and intraperitoneal administration of ED7 wasshown to reduce Adriamycin induced nephropathy in animals [114, 115,117], it is believed this is less of an issue as well. Third, as onlyED7 was tested as a model test agent representing activating antibodies,it could be reasoned that other activating antibodies can be moreefficacious biological agents in vivo. While not necessarily so (see thenext point), it is believed that future studies with additionalanti-CD11b activating antibodies would be a logical step to investigate'such a possibility. A limitation here is that there aren't very manyanti-CD11b/CD18 activating antibodies available for use in rodents, andthat they do not have cross-reactivity between species, thus limitingtheir validation in multiple species. Fourth, the full IgG molecule ofED7 was used in the studies presented here. Since the ED7 IgG would, inaddition to activating CD11b/CD18, dimerize CD11b/CD18 upon binding theintegrin on the leukocyte cell surface, it is also plausible that areason for the differences observed between the two types of agonistswas due to the IgG induced dimerization, suggesting that anon-dimerizing Fab fragment of the activating antibodies may be better.However, Lefort, et al. recently showed that non-dimerizingFab-fragments of activating anti-CD1b mAbs are sufficient to induceoutside-in signaling in cells, whereas the Fab of non-activating,blocking anti-CD11b mAb 44 are not [71]. They also showed thatCD11b/CD18 activation using activating mAbs, but not clustering, wassufficient to induce outside-in signaling in cells. Recent reports havesuggested that activating antibodies can induce global conformationalchanges in the integrin heterodimer and that such changes mimic ligandbinding induced outside-in signaling [71, 72]. It has also beenpreviously reported that constitutively active integrin mutants, wherethe ligand-binding DA-domain of CD11b is locked in its activeconformation, do not affect intracellular signaling [72]. Theseobservations and our data suggests that LA1 binding to the □A-domain ofCD11b may induce only local, domain-limited conformational changes inCD11b/CD18, whereas activating antibodies (whether IgG or Fab) andligands induce a more global conformational activation of the entireintegrin heterodimer and the concomitant outside-in signaling. It islikely that small molecule agonists, like LA1, prime integrin CD11b/CD18and induce intermediate affinity conformations [70]. This might berelevant in the setting of vascular repair where the findings hereinwould suggest that ED7-bound leukocytes could be more “pro-inflammatory”in nature and enhance disease as compared to the LA1-bound cells. Futurestructural and functional studies will further define the actualactivation state of LA1-bound CD11b/CD18. Thus, while it is plausiblethat Fab fragments of other biological agonists of CD11b/CD18 can showefficacy in a select few inflammatory models, it is also quite possiblethat anti-integrin activating antibodies in general, due to theirdisplay of some of the undesirable side-effects that have been describedfor the integrin antagonists [94-100], can have limited effectivenessand utility as a therapeutic. This shows that small molecules are bettersuited for the development of integrin agonists as therapeutics.

In conclusion, small molecule based agonists of CD11b/CD18 represent apreferred and effective pharmacological approach for the design of novelagents to treat a variety of inflammatory and autoimmune diseases inmammals.

EXAMPLE 2

TLR4-mediated signaling requires participation of the adaptor proteinMyD88 (43) and MyD88−/− mice are protected from kidney damage followingIRI. TLR4 activation leads to the binding and stabilization of adaptorprotein MyD88, which recruits downstream kinases to initiateNf-kB-mediated pro-inflammatory signaling. Subsequently, TLR4 signalinginduces a negative feedback loop by endogenous activation of CD11b/CD18,which activates Syk to phosphorylate MyD88, tagging it forubiquitin-mediated destruction. This shows that CD11b/CD18 agonists leadto accelerated degradation of MyD88, thereby inducing a faster dampeningof TLR4-mediated pro-inflammatory signaling pathways. A pilot experimentwas carried out to validate this hypothesis by determining the levels ofMyD88 in human monocytic THP-1 cells. It was found that TLR4 agonist LPSproduced a robust MyD88 signal that was stable for at least 4 hours(FIG. 1). However, co-treatment of cells with LA1 lead to a much fasterdegradation of MyD88. Indeed, incubation of cells with LA1 alone (in theabsence of LPS) resulted in a complete degradation of MyD88 in less than2 hours, showing that activation of CD11b/CD18 can down-modulateMyD88-dependent intracellular signaling in leukocytes. This supports thestatements that leukadherins mediated priming of integrins foractivation negatively regulates intracellular signaling, including theinflammatory NF-kB signaling. Additionally, LA1 treatment of cellsresulted in induction of Syk phosphorylation in leukocytes by Westernblot based analysis of pSyk (not shown). Leukocytes were incubated withLA1 for 0-60 minutes and the levels of phosphorylated Syk, total Syk andGAPDH were assayed, showing that LA1 induces Syk phosphorylation.

EXAMPLE 3

In this experiment, four groups of tumor-bearing animals were used(n=-16/group) and tumor growth is shown in FIG. 2A: 1) Treatment withvehicle alone (control, blue line), 2) Treatment with leukadherin LA1alone (green line), 3) Treatment with taxol alone (purple line) and 4)Co-treatment with LA1 and Taxol (red line). The syngeneic murine mammaryadenocarcinoma cell line Cl66 (moderately metastatic and is related tothe highly metastatic 4T1 cell line) was orthotopically introduced inBalb/c animals, by injecting ˜5×105 Cl66 cells under the left mammaryfat pad of WT BALB/c mice (approx. 8-10 weeks old). The tumor size wasmeasured using calipers every other day until the end of the experiment(approx. 5 weeks). 7-days post implantation, the tumors in all animalsbecame palpable, with an average size of ˜100 mm3. At that time, animalsin each group were treated as described. LA1 was administered daily forthe first two weeks and then every other day until the end of theexperiment. Animals in Group 1 received Saline injections, as control.The results in this figure show a significant reduction in the rate oftumor growth in Taxol and, surprisingly, in LA1 treated animals (FIG.2C, control group is shown in FIG. 2B). Additionally, and similarlysurprisingly, animals co-treated with the compound LA1 and Taxol showedthe highest reduction in the rate of tumor growth. This shows thatleukadherins-mediated reduction in inflammatory leukocytes cansignificantly reduce tumor growth. It can also enhance the efficacy oftraditional chemotherapy in various cancers, including breast cancer, toproduce a synergistic response to the treatment. It can also reduce theincidence and the size of metastases.

EXAMPLE 4

The efficacy of LA1 was tested in an experimental model of Diabeticnephropathy (DN), where inflammation plays a major role. It was surmisedthat reducing leukocyte activation, recruitment and influx can be abeneficial strategy for developing therapeutics against DN. It was foundthat LA1 significantly prevents and/or treats DN, as seen in a murinemodel, which has been shown to more closely mimic the human disease (theBTBR ob/ob mouse model). Daily administration of our compoundsignificantly reduced the number of infiltrating leukocytes in thekidney and preserved kidney function in fully diabetic animals. LA1reduced glomerular damage and glomerular mesangial sclerosis. Data isshown in FIG. 3.

Additionally, in a model of allograft transplantation, co-treatment ofanimals with LA1 (with cyclosporine) provided much better engraftment ofthe donated kidney, suggesting that LA1 has therapeutic value forallograft nephropathy and other similar transplants (data not shown).

EXAMPLE 5

It was shown that β2 integrin agonist leukdherin LA1 modulates theexpression levels of various pro-inflammatory factors in cells. Humanmacrophages were treated with LPS in the absence or presence of LA1 invitro and determined the levels of mRNA in the cells at various timepoints using Nanostring inflammation array (184 genes) and exiqonmicroRNA panels. The data shows that mRNA levels (and thus expression)of a number of pro-inflammatory factors (FIGS. 15A-15G and FIGS.16A-16G, list in FIG. 17), that are upregulated by LPS treatment ofthese cells is significantly reduced in cells co-treated with LA1.

This supports the finding that leukadherins mediated priming ofintegrins for activation negatively regulates intracellular signaling,including the inflammatory NF-kB signaling.

EXAMPLE 6

FIGS. 18A-18B and FIGS. 19A-19B show adhesive behavior of vehicle-, Mn2+or LA-treated various CD11b/CD18 transfectant cells under the wall shearstress. FIGS. 18A-18B show cells resistant to detachment in shear flow.The total number of cells remaining bound at the end of each shearstress were plotted Error bars are ±s:d: (n=3).*: P<0.05; **: P<0.01;***: P<0.001; and ns, not significant. In FIGS. 19A-19B, error bars are±s:d: (n=3).*: P<0.05; **: P<0.01; ***: P<0.001 ; and ns, notsignificant. The results show that LA1, unlike Mn2+, induces priming ofCD11b/CD18 and converts it into intermediate conformation.

EXAMPLE 7

LA1 is non-toxic in vivo. A subacute toxicity assessment of LA1 in ratswas performed. IP administered LA1 (˜3 mg/kg/d for 21 d) failed toinduce any overt toxicity or mortality in SD rats of both sexes. FIG. 20shows that the biochemical measurements in serum and liver ofLA1-treated rats revealed no appreciable changes in enzyme levels orserum constituents, such as proteins, cholesterol, urea and creatinine.Haematological constants in LA1-treated rats were on par with those ofcontrols.

LA1 had no effect on the daily food intake or growth. Autopsy revealedno alterations in relative organ weights of various vital organs (lung,heart, spleen, liver, and kidney) or their histoarchitecture (FIG. 21).This shows that LA1 does not produce any significant acute andcumulative toxicity at the doses administered.

EXAMPLE 7

FIGS. 22A-22E show that leukadherins are harmless to endothelial cellsand are confocal images of DAPI-stained Human Umbilical Vein EndothelialCells (HUVECs, blue) showing that LA1-mediated adhesion of neutrophilsdoes not increase HUVEC apoptosis as measured by the Terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, whereincorporation of labeled dUTP marks apoptotic cells (Green). It showsthat while positive control cells show a lot of TUNEL staining (green),LA1 treated cells do not. Leukadherins only temporarily promote thenatural interaction between inflamed or denuded endothelium andleukocytes, but not to “healthy endothelium” (as the healthy cells donot express CD11b/CD18 ligands, such as ICAM-1 and CD40). In support ofthis argument, neither we nor others studying knock-in animals thatexpress constitutively active mutants of integrins LFA-1 [54, 55] and□4□7 [56] have observed any signs of vascular injury in any experimentalmodel. FIGS. 22A-22E show an absence of apoptosis in co-cultures of LA-1activated leukocytes and HUVEC cells.

FIG. 23 shows leukadherin LA1 concentration in mouse blood over timeafter administration via two different routes, showing that it isbioavailable in animals. With both oral (PO) and intraperitoneal (IP)dosing of LA1 at 10 mg/kg into animals (mice) showed high concentrationsof LA1 in plasma (as measured by LC-MS), showing that LA1 isbioavailable and has micromolar levels in blood with mean residence timeof at least a few hours.

FIGS. 24A-24B show that leukadherin LA1 reduces the rate of tumorre-growth upon treatment. It shows (24A) the rate of tumor growth inanimals in the various groups as well as (24B) the relative tumor volumein animals at the end of the study. Data shown are means±SEM. *, P<0.05;**, P<0.001 (by one-way ANOVA). In this experiment, four groups oftumor-bearing animals were used (n=11/group): 1) Treatment with salinealone (red line), 2) Treatment with leukadherins LA1 alone (orangeline), 3) Treatment with a single dose of 20 Gy irradiation (two 10%+10%vertices) alone (black line) and 4) Treatment with a single dose of 20Gy irradiation and then daily LA1 (at −2 hours) (blue line). Thesyngeneic murine mammary adenocarcinoma cell line Cl66 (moderatelymetastatic and is related to the highly metastatic 4T1 cell line) wasorthotopically introduced in Balb/c animals, by injecting approximately5×105 Cl66 cells under the left mammary fat pad of WT BALB/c mice(approximately 10 weeks old). The tumor size was measured using calipersevery other day until the end of the experiment (approximately 5 weeks).7-days post implantation, the tumors in all animals became palpable,with an average size of ˜100 mm3. At that time, animals in groups 3 and4 were treated with irradiation. Animals in group 4 received LA1,starting at 2 hours prior to irradiation. LA1 was administered daily forthe first week and then every other day until the end of the experiment(when the tumor size reached 10% of the body weight). Animals in Group 1received Saline injections, as control. The results in FIGS. 24A-24Bshow a significant reduction in the rate of tumor growthpost-irradiation in LA1 treated animals. Additionally, and quitesurprisingly, animals treated with the compound LA1 alone, in theabsence of any irradiation (Group 2) also showed a reduced rate of tumorgrowth, to the level similar to the rate that was observed withirradiation alone.

FIG. 25 shows a survival curve showing that treatment of animals withleukadherin LA1, with or without sublethal total body irradiation, doesnot negatively affect animal mortality. Control groups (16 mice pergroup) of 6-8 weeks old C57BL/6J mice received a single sublethal dose(6 Gy) of total body radiation (TBI) and were monitored for survivalwith radiation alone. The experimental groups (16 mice per group) of 6-8weeks old C57BL/6J received a single sublethal dose (6 Gy) of total bodyradiation (TBI) and were administered leukadherin LA1 (20 μg/animal) forseven consecutive days. Additionally, two additional control groups ofanimals were monitored—animals with no treatment at all or animals thatwere administered LA1 for seven consecutive days in the absence of anyirradiation. The results, shown in FIG. 25, demonstrate that treatmentof LA1 did not result in any increase in animal mortality overLA1-untreated animals. In fact, LA1 treatment shows a protective effect,showing an optimized dose of LA1 would be even more protective.

FIGS. 26A-26C show the analysis of hematopoiesis and HSC compartment inLA1 (Red bars), Vehicle control (Blue bars) treated groups of mice at 4weeks after 6 Gy of total body irradiation (TBI), showing that LA1 ishighly radio-protective and a radio-mitigator. FIGS. 26A-26C show thatLA1 protects hematopoiesis and HSC compartments and cells aftersublethal IR even when the LA1 treatment is delayed, thus effectivelymitigating the adverse effects of sublethal IR on the hematopoieticsystem and HSCs. Mice were exposed to 6 Gy of TBI (at 0.5 Gy/min), andtreated intraperitoneally (i.p.) with LA1 (1 mg/kg) or vehicle alone for7 consecutive days starting at 24 hours post TBI. Analysis of HSCs4-weeks post TBI showed a significant improvement in the LA1 treatedgroup, as measured by BM cellularity, the frequency of LKS+ BM cells andLKS+ CD150+ CD48− BM cells (highly enriched for LTR-HSCs). The sametrend was observed at 8 and 12 weeks after TBI (data not shown),indicating that delayed treatment with LA1 accelerates recovery ofhematopoiesis and either preserves HSCs or facilitates the recovery ofHSC compartment after sublethal IR.

FIG. 27 shows LA1 dose-dependently reduces T-cell proliferation, asmeasured by a Mixed lymphocyte reaction (MLR). Additionally, LA1decreased IFN-□ production by T-cells in an antigen dose-dependentfashion (MOG-peptide, data not shown) when antigen-reactive T cells fromthe draining lymph nodes of mice were assessed.

FIG. 28 shows that LA1 treatment of human macrophages from lupuspatients with dsRNA (a lupus antigen) significantly reduced the TNF-αrelease, whereas exposure of macrophages to LA1 alone had no effect oncellular morphology and LA1 alone did not induce TNF-α over 24 hrs. Thisalso shows that LA1 significantly attenuates pro-inflammatory phenotypein macrophages and neutrophils from lupus patients, including those fromsubjects with the coding variant of R77H.

FIG. 29 shows that LA1 induces Syk phosphorylation in leukocytes byWestern blot based analysis of Syk phosphorylation. Leukocytes wereincubated with LA1 for 0-60 minutes and the levels of phosphorylatedSyk, total Syk and GAPDH were assayed, showing that LA1 induces Sykphosphorylation.

Results

LA1 is non-toxic in viva A pilot in vitro ADME assay (neutrophils,hepatocytes, hERG) showed no adverse findings with LA1 (at up to 100 □M)(not shown). Next, a subacute toxicity assessment was performed of LA1in rats in the Comparative Pathology core. IP administered LA1 (˜3mg/kg/d for 21 d) failed to induce any overt toxicity or mortality in SDrats of both sexes. Further, no significant alterations either inrelative organ weights or their histology were discernible at terminalautopsy. LA1 had no effect on the daily food intake or growth. Autopsyrevealed no alterations in relative organ weights of various vitalorgans (lung, heart, spleen, liver, and kidney) or theirhistoarchitecture. Haematological constants in LA1-treated rats were onpar with those of controls. The biochemical measurements in serum andliver of LA1-treated rats revealed no appreciable changes in enzymelevels or serum constituents, such as proteins, cholesterol, urea andcreatinine. This shows that LA1 does not produce any significant acuteand cumulative toxicity at the doses administered.

Pilot oral vehicle evaluation. Five different salts of LA1 (Na, K, NH4,Ca and Mg) were characterized using XPRD, DCS and TGA and tested foraqueous solubility (not shown), which showed improved crystalline formfor LA1 in its magnesium salt, although poor equilibrium aqueoussolubility (˜0.6 μg/ml). Additionally, eight different vehicleformulations were prepared using the Mg-salt of LA1, usingmethylcellulose (MC), MC/SDS mixture, Tween, ETPGS, Captisol, sucrose,gelatin or propylene glycol. 10 mg/mL slurry samples of LA1 wereprepared in each of the eight formulations and LA1's solubility wasexamined using LC-MS. At least three—10% Tween80, 20% ETPGS and 30%Captisol—showed enhanced solubility of LA1 (3-4 mg/mL). Subsequently,re-dispersability of LA1 was tested using two simulated biologicalfluids—simulated gastric fluid (SGF) and Fasted State Simulated GastricFluid (FaSSIF). Results showed that LA1 had much higher solubility ineach of the three formulations, up to ˜0.15 mg/mL in the case of 10%Tween80, showing that such vehicle screening approaches have thepotential to significantly improve delivery and dosing of LA1 toanimals.

A syngeneic xenograft model was used to address the effects ofinflammatory cell recruitment on tumor cell re-growth afterradio-therapy. In this pilot experiment, it was decided to use fourgroups of tumor-bearing animals (n=11/group): 1) Treatment with salinealone, 2) Treatment with leukadherins LA1 alone, 3) Treatment with asingle dose of 20 Gy irradiation (using the LATTICE method, two 10%+10%vertices) alone and 4) Treatment with a single dose of 20 Gy irradiationand LA1 (at −2 h). The murine mammary adenocarcinoma cell line Cl66(moderately metastatic and is related to the highly metastatic 4T1 cellline) was orthotopically introduced in all animals, by injecting ˜5×105Cl66 cells under the left mammary fat pad of WT BALB/c mice (10 weeksold). The tumor size was measured using calipers every other day untilthe end of the experiment (approximately 5 weeks). 7-days postimplantation, the tumors, in all animals became palpable, with anaverage size of ˜100 mm³. At that time, animals in groups 3 and 4 weretreated with irradiation. Animals in group 4 received LA1, starting at 2hours prior to irradiation. LA1 was administered daily for the firstweek and then every other day until the end of the experiment (when thetumor size reached 10% of the body weight). Animals in Group 1 receivedSaline injections, as control. Preliminary results of this experimentare presented in FIGS. 24A-24B. They show a significant reduction in therate of tumor growth post-irradiation in LA1 treated animals.Additionally, and quite surprisingly, animals treated with the compoundLA1 alone, in the absence of any irradiation (Group 2) also showed areduced rate of tumor growth, to the level similar to the rate that wasobserved with irradiation alone.

Treatment of human macrophages (as well as dendritic cells andneutrophils, not shown) from normal healthy volunteers and from lupuspatients with ds RNA antigen (R848) significantly stimulated TNF-αrelease compared with macrophages alone, as shown in FIG. 28 (n=14).When cells were exposed to R848 and LA1, their ability to release TNF-αwas significantly impaired (p=0.05). Note that exposure of cells to LA1alone had no effect on cellular morphology and LA1 alone did not induceTNF-α over 24 hrs. Similar results were obtained with FMLP and otherreagents. Thus, the compounds of the present invention significantlyattenuate pro-inflammatory phenotype in leukocytes (such as macrophages,dendritic cells and neutrophils) from patients, including those fromsubjects with the coding variant of CD11b, such as the R77H variant.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology, which has been used is intended tobe in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventioncan be practiced otherwise than as specifically described.

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1-35. (canceled)
 36. A method for treating diabetic nephropathy, themethod comprising: administering to a patient in need thereof a β2integrin agonist having the formula

or a pharmaceutically acceptable salt thereof, wherein: A is absent oris selected from alkyl and alkenyl, B is absent or is selected fromalkyl, alkenyl, O, S, and NR⁴, X is selected from O and S, R¹ isselected from alkoxycarbonyl, aryl, heteroaryl, carbocyclyl, andheterocyclyl, R² is H, R³ is phenyl which is unsubstituted orsubstituted with one or two substituents independently selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, alkyl, andcarboxy, and R⁴ is selected from hydrogen and alkyl; and improving thehealth of the patient's kidneys.
 37. The method of claim 36, wherein theβ2 integrin agonist is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 38. The method of claim37, wherein the β2 integrin agonist is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 39. The method of claim36, wherein the β2 integrin agonist is:

or a pharmaceutically acceptable salt thereof.
 40. The method of claim36, wherein the β2 integrin agonist is:

or a pharmaceutically acceptable salt thereof.
 41. The method of claim36, wherein the β2 integrin agonist is:

or a pharmaceutically acceptable salt thereof.
 42. The method of claim36, wherein the β2 integrin agonist is:

or a pharmaceutically acceptable salt thereof.
 43. The method of claim36, wherein the β2 integrin agonist is:

or a pharmaceutically acceptable salt thereof.